Development of Immunoassays for Type II Synthetic Pyrethroids. 1. Hapten Design and Application to Heterologous and Homologous Assays

Article abstract of DOI:10.1021/jf970438r

Immunoassays differing in selectivities for pyrethroid insecticides have been developed for the detection of type II pyrethroids, including deltamethrin, cypermethrin, and λ-cyhalothrin. Two approaches were employed in hapten synthesis to raise antibodies with different cross-reactions: (1) use of three spacer attachment points to offset different parts of molecules from the points of attachment and (2) use of linkers with and without bulky groups in the enzyme conjugate to reduce antibody affinities for the spacer arm in the immunoassay. The first approach resulted in the preparation of three series of haptens with a spacer attached (1) at the aromatic moiety of pyrethroid, (2) through the middle of the molecule, and (3) at the cyclopropane moiety. Haptens based on the derivatives of the pyrethroid metabolites were also prepared. The second approach involved the use of a linker with a bulky (cyclohexane ring) functionality for preparation of an enzyme conjugate. While most combinations of antibody and conjugate could be used in immunoassays for detection of deltamethrin in the 10-100 μg/L range, in most cases the limits of detection of the assays (for total isomers of a particular target pyrethroid) were lowered 10-50 fold by treatment of the pyrethroid standards with dilute alkali to produce a different isomer mix. Fifteen antisera prepared using 8 haptens were each screened with 14 peroxidase conjugates, and 26 antibody/conjugate combinations were selected for further study on the basis of the assay sensitivity, dynamic behavior, and specificity for deltamethrin, cypermethrin, and cyhalothrin. These immunoassays provided 50% inhibition of antibody binding (IC₅₀) values between 1.5 and 4.2 μg/L of isomerized total deltamethrin and limits of detection of 0.2-0.7 μg/L. The most sensitive immunoassay for total deltamethrin was obtained using cypermethric acid-KLH as the immunogen and a conjugate based on a derivative of cypermethrin coupled through the middle of the molecule to peroxidase. These provided an IC₅₀ of 2 μg/L and a limit of detection of 0.2 μg/L of isomerized total deltamethrin. However, no particular hapten design produced antisera of clearly superior sensitivity or specificity for deltamethrin. Differing cross-reactions with the closely related pyrethroids, deltamethrin, cypermethrin, and cyhalothrin, were obtained, and for several antibodies the cross-reaction as well as the limits of detection could be altered by varying the conjugate combinations. Each of the 12 antibody/enzyme conjugate combinations that sensitively detected deltamethrin were very stereospecific, detecting the αS, 1R cis, (DM1), and αR, 1R cis (DM2) isomers only; the assay sensitivity was greater for the latter isomer.

Full text of DOI:10.1021/jf970438r

520
J. Agric. Food Chem. 1998, 46, 520−534
Develop m en t of Im m u n oa ssa ys for Typ e II Syn th etic P yr eth r oid s. 1.
Ha p ten Design a n d Ap p lica tion to Heter ologou s a n d Hom ologou s
Assa ys
Nanju Lee,† David P. McAdam, and J ohn H. Skerritt*
CSIRO Plant Industry, North Ryde, NSW 2113, and Canberra, ACT 2601, Australia
Immunoassays differing in selectivities for pyrethroid insecticides have been developed for the
detection of type II pyrethroids, including deltamethrin, cypermethrin, and λ-cyhalothrin. Two
approaches were employed in hapten synthesis to raise antibodies with different cross-reactions:
(1) use of three spacer attachment points to offset different parts of molecules from the points of
attachment and (2) use of linkers with and without bulky groups in the enzyme conjugate to reduce
antibody affinities for the spacer arm in the immunoassay. The first approach resulted in the
preparation of three series of haptens with a spacer attached (1) at the aromatic moiety of pyrethroid,
(2) through the middle of the molecule, and (3) at the cyclopropane moiety. Haptens based on the
derivatives of the pyrethroid metabolites were also prepared. The second approach involved the
use of a linker with a bulky (cyclohexane ring) functionality for preparation of an enzyme conjugate.
While most combinations of antibody and conjugate could be used in immunoassays for detection of
deltamethrin in the 10-100 µg/L range, in most cases the limits of detection of the assays (for total
isomers of a particular target pyrethroid) were lowered 10-50 fold by treatment of the pyrethroid
standards with dilute alkali to produce a different isomer mix. Fifteen antisera prepared using 8
haptens were each screened with 14 peroxidase conjugates, and 26 antibody/conjugate combinations
were selected for further study on the basis of the assay sensitivity, dynamic behavior, and specificity
for deltamethrin, cypermethrin, and cyhalothrin. These immunoassays provided 50% inhibition of
antibody binding (IC50) values between 1.5 and 4.2 µg/L of isomerized total deltamethrin and limits
of detection of 0.2-0.7 µg/L. The most sensitive immunoassay for total deltamethrin was obtained
using cypermethric acid-KLH as the immunogen and a conjugate based on a derivative of
cypermethrin coupled through the middle of the molecule to peroxidase. These provided an IC50 of
2 µg/L and a limit of detection of 0.2 µg/L of isomerized total deltamethrin. However, no particular
hapten design produced antisera of clearly superior sensitivity or specificity for deltamethrin.
Differing cross-reactions with the closely related pyrethroids, deltamethrin, cypermethrin, and
cyhalothrin, were obtained, and for several antibodies the cross-reaction as well as the limits of
detection could be altered by varying the conjugate combinations. Each of the 12 antibody/enzyme
conjugate combinations that sensitively detected deltamethrin were very stereospecific, detecting
the RS, 1R cis, (DM1), and RR, 1R cis (DM2) isomers only; the assay sensitivity was greater for the
latter isomer.
Keyw or d s: Synthetic pyrethroid; deltamethrin; cypermethrin; cyhalothrin; hapten; immunoassay;
agrochemical
INTRODUCTION
range of crops, including potatoes, sugar beet, cereals,
coffee, tea, rice, vines, tobacco, and cotton for control of
a wide range of pests, including Helicoverpa spp. Pyre-
throids are also used in protection of stored commodities
and for the control of lice, fleas, ticks, and scabies in
domestic and farm animals. The increasing use of
synthetic pyrethroids, compared with that of other
classes of insecticides, is attributed to their remarkably
high insecticidal activities and low toxicities to mam-
mals (Elliott, 1977; Davies, 1985).
The synthetic pyrethroids and natural pyrethrins can
be divided into two groups of compounds on the basis
of chemical structure and mechanism of action at insect
target sites. The type I compounds are simple cyclic
alcohol esters of 2,2-dimethyl-3-(2-methyl-1-propenyl)-
cyclopropanecarboxylic acid. The type II compounds are
esters of an arylcyanohydrin. The type I pyrethroids
are less photostable than the type II pyrethroids,
restricting their use to indoor application (Davies, 1985),
Synthetic pyrethroids are used widely in domestic,
public health, agricultural, forestry, and veterinary
applications (Hassall, 1990). In domestic use, pyre-
throids are used to control cockroaches, houseflies, and
many other household insects. In public health, delta-
methrin, cypermethrin, fenpropathrin, fenvalerate, and
permethrin are used against mosquitoes, simulid, and
tse-tse fly. In forestry, permethrin is used to control
spruce budworm in the pine forest ecosystem. In
agricultural situations, pyrethroids are used on a wide
* Address correspondence to this author at CSIRO Plant
Industry, GPO Box 1600, Canberra, ACT 2601, Australia
(telephone +61 2 6246 5350; fax +61 2 6246 5351; e-mail
J .Skerritt@pi.csiro.au).
† Present address: U.S. Department of Agriculture, Food
Animal Protection Research Laboratory, College Station, TX
77845.
S0021-8561(97)00438-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/14/1998

Hapten Design for Pyrethroid Immunoassays
J. Agric. Food Chem., Vol. 46, No. 2, 1998 521
have different toxicological effects, and may have slightly
different mechanisms of action at the insect neuron
(Hassall, 1990). Residues of synthetic pyrethroids can
be analyzed by either high-performance liquid chroma-
tography (HPLC) or gas-liquid chromatography (GLC).
The synthetic pyrethroids exist as either a single
stereoisomer (e.g. deltamethrin) or a mixture of stere-
oisomers and can undergo transformations to insecti-
cidally inactive isomers in water (Takahashi et al., 1985;
Maguire, 1990). Analysis of the optical isomers of a
variety of pyrethroids, including λ-cyhalothrin, cyper-
methrin and R-cypermethrin, has been reported using
normal-phase HPLC and Pirkle columns (Cayley and
Simpson, 1986; Lisseter and Hambling, 1991; Perschke
and Hussain, 1992). Pyrethroids containing halogen
atoms, such as deltamethrin, cypermethrin, cyhalothrin,
and bifenthrin, are commonly analyzed by GLC with
electron capture detection. The sensitivities for these
pyrethroids are comparably lower than those of other
pesticides containing halogen groups, such as orga-
nochlorine insecticides, with limits of detection between
0.01 and 0.05 mg/kg (Chapman and Harris, 1978). The
sensitivities for non-halogen-containing pyrethroids are
even lower.
approaches to hapten production and the antibodies that
resulted from these synthetic approaches were evalu-
ated.
MATERIALS AND METHODS
Ma ter ia ls. Chemicals used in hapten synthesis were
obtained from either Aldrich (Milwaukee, WI) or Sigma (St.
Louis, MO). Other chemicals used in preparation of buffers
were purchased from either Ajax Chemicals (Sydney, Austra-
lia) or BDH-Merck (Melbourne, Australia). Silica gel 60 (70-
230) mesh was purchased from Merck (Darmstadt, Germany).
Bovine serum albumin (BSA) and horseradish peroxidase
(HRP) were purchased from Boeheringer Mannheim, Ger-
many. Keyhole limpet hemocyanin (KLH) and the BCA
protein assay were purchased from Pierce (Rockville, IL).
Tween 20 and Freund’s complete and incomplete adjuvants
and 3,3′,5,5′-tetramethylbenzidine were obtained from Sigma.
Econo-Pac 10DG columns were obtained from Bio-Rad (Her-
cules, CA). Protein A agarose was purchased from Phamacia
(Uppsala, Sweden). Maxisorp polystyrene 96-well plates were
obtained from Nunc (Roskilde, Denmark), and swine anti-
rabbit IgG was purchased from Dako (Glostrup, Denmark).
Deltamethrin isomers (DM1, DM2, DM3, DM4, DM1′, DM2′,
DM3′, and DM4′) were gifts from AgrEvo Environmental
Health (Berkamsted, U.K.). Deltamethrin [(S)-R-cyano-3-
phenoxybenzyl (1R)-cis-3-(2,2-dibromovinyl)-2,2-dimethylcy-
clopropanecarboxylate], λ-cyhalothrin [(RS)-R-cyano-3-phenox-
ybenzyl (Z)-(1RS)-cis-3-(2-chloro-3,3,3-trifluoropropenyl)-2,2-
dimethylcyclopropanecarboxylate], and R-cypermethrin [(RS)-
R-cyano-3-phenoxybenzyl (1RS)-cis-3-(2,2-dichlorovinyl)-1,1-
dimethylcyclopropanecarboxylate] were provided by the
Australian Government Analytical Laboratory (Sydney).
Ch em ica l An a lyses. 1H and 13C NMR spectra were
recorded with a Varian Gemini II instrument, operating at
300 and 75 MHz, respectively, using deuterated chloroform
(CDCl3, 99.8% D) as solvent with 0.03% (v/v) tetramethylsilane
(Aldrich) as internal standard. Thin-layer chromatography
(TLC) was performed on 0.2 mm thick precoated silica gel 60
F254 on plastic sheets from Merck. TLC spots were detected
by irradiation with UV light and by exposure to iodine vapor.
If necessary, TLC plates were sprayed with either 0.03%
KMnO4 in concentrated H2SO4 (caution) or 1% (w/v) silver
nitrate. Column chromatography was performed on silica gel
(particle size 63-200 µm, 70-230 mesh ASTM), Merck).
Flash chromatography (J . T. Baker, Philipsburg, NJ ) was
performed using silica gel 200-400 mesh (Aldrich). Radial
chromatography on a Chromatotron (Harrison Research, Palo
Alto, CA) was performed for small samples (<200 mg) on 2-
and 4-mm-thick silica gel (gel 60 PF254 containing gypsum,
Merck) coated plates.
Ha p ten Syn th esis. The first series of haptens with a
spacer attachment at the aromatic moiety was prepared by
an esterification of 2-hydroxydeltamethrin/4-hydroxydelta-
methrin with succinic anhydride (Zhang and Scott, 1994). The
second series of haptens involved a conversion of an R-cyano
group of the pyrethroid to a corresponding acid for a spacer
attachment (Figure 1). The third series of haptens involved
an oxidation of the vinyl moiety using KIO3/KMnO4 to produce
an acid, followed by a reaction with tert-butyl 3-aminopro-
panoate (Figure 2). In addition, haptens were prepared that
derived from pyrethroid metabolites including deltamethric
acid, cypermethrin acid, cyhalothric acid, and 3-phenoxyben-
zaldehyde cyanohydrin. Use of a bulky functional group in a
spacer in the second approach was expected to reduce the
affinity of the antibodies for the spacer in immunoassays and
hence increase the sensitivity of antibody for free analyte in
immunoassay (Brady et al., 1989; Hill et al., 1993). The bulky
spacer was prepared using a method by Hill et al. (1993) and
was introduced only for those haptens for which enzyme
conjugates with linear spacers exhibited moderate sensitivity
(IC50 of 10-100 µg/L) in competition immunoassay.
These problems have stimulated research on alterna-
tive techniques for pyrethroid residue analysis, such as
immunoassay. Few immunoassays have been developed
that detect type II synthetic pyrethroids. An immu-
noassay based on a monoclonal antibody to (1R)-trans-
permethric acid coupled to protein (Bonwick et al., 1994;
Pullen and Hock, 1995a,b) was able to detect the type I
pyrethroids such as allethrin, bioallethrin, S-bioal-
lethrin, permethrin, bioresmethrin, and pyrethrin, while
an assay based on a polyclonal antibody enabled the
detection of both type I (permethrin and S-bioallethrin)
and II pyrethroids (cyfluthrin and cypermethrin), al-
though other type II pyrethroids such as deltamethrin
were not detected. The primary objective for this work
was to develop two types of immunoassays, namely a
class-specific assay (detecting type II pyrethroids) and
compound-selective assays (detecting either delta-
methrin, cypermethrin, or λ-cyhalothrin). These com-
pounds were the targets for immunoassay development
in this study as they comprise ∼81% of total pyrethroids
used in the Australian cotton industry for control of
Helicoverpa spp. (Barrett et al., 1991). Apart from
having differing mixtures of isomers in their insecticidal
formulations (Hassall, 1990), they differ only in the
halogen atoms or groups on the vinyl side chain:
deltamethrin has two bromine atoms, cypermethrin has
two chlorine atoms, and cyhalothrin has a chlorine atom
and a trifluoromethyl group. Pyrethroids are relatively
large molecules with no obvious functional group for
direct conjugation. Previous work demonstrated that
a linker could be placed at the aromatic moiety (De-
moute et al., 1986) and at the cyclopropane moiety
(Stanker et al., 1989; Hill et al., 1993). Haptens derived
from the pyrethroid metabolites also have been prepared
(Pullen and Hock, 1995a,b; Wengatz et al., 1996). These
immunoassays were selective for compounds containing
the dimethyl or (1R)-trans-dichlorovinyl dimethylcyclo-
propane moiety, because they were based on one hapten
derived from trans-permethric acid. Another possible
place for coupling would be at the cyano group of the
type II pyrethroid. In this study, a range of synthetic
Cou p lin g a t th e Ar om a tic Moiety. (S)-R-Cyano-[3′-(2′′-
hydroxyphenoxy)phenyl]methyl (1R,3R)-3-(2′,2′-Dibromoethen-

522 J. Agric. Food Chem., Vol. 46, No. 2, 1998
Lee et al.
F igu r e 2. Reaction scheme for synthesis of haptens with a
spacer attachment at the cyclopropane moiety. Other details
are as for legend to Figure 1.
ethyl acetate (6:1)] to yield 223 mg of 1 (4.3%) and 510 mg of
2 (10%) as bright yellow oils. The identities of the compounds
were confirmed by comparison with Rf values [for n-hexane/
1
ethyl acetate (1:1)] and H NMR [(CD3)2CO] data of Zhang and
Scott (1994).
(S)-R-Cyano-[3′-(2′′-(2-carboxyethylcarbonyloxy)phenoxy)-
phenyl]methyl (1R,3R)-3-(2′,2′-Dibromoethenyl)-2,2-dimethyl-
cyclopropanecarboxylate (3). Succinic anhydride (51 mg, 0.51
mmol) and dimethylaminopyridine (DMAP, 2 mg) were added
to the alcohol 1 (223 mg, 0.43 mmol) in dry pyridine (2 mL),
and the mixture was stirred overnight. The solution was
extracted with ethyl acetate (30 mL), and the resulting organic
layer was washed with 1.0 M HCl, water, and brine and then
dried. The organic solution was concentrated and the residue
was chromatographed on silica using a Chromatotron [ethyl
acetate/petroleum ether (bp 60-80 °C, 4:6] to give the product
as a pale yellow oil (41.3 mg, 28.9%): TLC Rf 0.21 [ethyl
1
acetate/petroleum ether 4:6]; H NMR (CDCl3) δ 7.0-7.5 (m,
aromatic H), 6.70 (d, dCH, J ) 8.5 Hz), 6.35 (s, CHCN), 2.67
(m, 12 lines, A2B2), 2.07 (t, CHC, J ) 8.5 Hz), 1.92 (d, CHCO,
J ) 8.5 Hz), 1.23 (s, CH3), and 1.19 (s, CH3).
F igu r e 1. Reaction scheme for synthesis of haptens with
spacer arm attachment at the R-position of benzylic carbon
for conjugation through the middle of the molecule. Refer to
Materials and Methods for compound names.
(S)-R-Cyano-[3′-(4′′-(2-carboxyethylcarbonyloxy)phenoxy)-
phenyl]methyl (1R,3R)-3-(2′,2′-Dibromoethenyl)-2,2-dimethyl-
cyclopropanecarboxylate (4). Succinic anhydride (23 mg, 0.23
mmol) followed by DMAP (2 mg) was added to the alcohol 2
(100 mg, 0.19 mmol) in dry pyridine (2 mL) and stirred
overnight. The solution was extracted in the same manner
as for 1, and the residue was chromatographed on silica using
a Chromatotron [ethyl acetate/petroleum ether (3:7)] to yield
a pale yellow oil (42.4 mg, 13.4%): TLC Rf 0.24 [ethyl acetate/
petroleum ether (3:7)]; 1H NMR (CDCl3) δ 7.0-7.4 (m, aromatic
H), 6.73 (d, dCH, J ) 8.3 Hz), 6.38 (s, CHCN), 2.88 (d, CH2-
CO, J ) 6.4 Hz), 2.84 (d, CH2CO, J ) 6.4 Hz), 2.10 (t, CHC, J
) 8.2 Hz), 1.94 (d, CHCO, J ) 8.6 Hz), 1.27 (s, CH3), and 1.23
(s, CH3).
(S)-R-Cyano-[3′-(2′′-(2-carboxyethylcarbonyloxy)phenoxy)-
phenyl]methyl (1R,3R)-3-(2′,2′-Dibromoethenyl)-2,2-dimethyl-
cyclopropanecarboxy Succinimidyl Ester. To the acid 3 (41.3
mg, 0.064 mmol) in dichloromethane (5 mL, 0 °C) was added
dicyclohexylcarbodiimide (DCC; 14.6 mg, 0.07 mmol) and
DMAP (5 mg) followed by N-hydroxysuccinimide (NHS; 7.4 mg,
0.07 mmol). The mixture was stirred overnight and then
filtered through Celite, and the solvent was evaporated to give
the active ester as a pale yellow oil.
yl)-2,2-dimethylcyclopropanecarboxylate (2-Hydroxydeltamethrin,
1) and (S)-R-Cyano-[3′-(4′′-hydroxyphenoxy)phenyl]meth-
yl (1R,3R)-3-(2′,2′-Dibromoethenyl)-2,2-dimethylcyclopropane-
carboxylate (4-Hydroxydeltamethrin, 2). Lead(IV) trifluoro-
acetic acid was prepared according to the method of Partch
(1967). Lead oxide (7.5 g, 0.011 mol) was added to a flask
containing trifluoroacetic acid (TFA, 17.6 mL) and trifluoro-
acetic acid anhydride (10 mL, caution), and the mixture was
stirred under nitrogen until a white crystalline compound was
formed. Deltamethrin (5.05 g, 10 mmol) was added slowly to
a flask containing lead (IV) trifluoroacetic acid (5.59 g, 10
mmol) and TFA (33.3 mL) in an ice bath, and the resultant
mixture stirred for 2 h. Then 10% K2CO3 (250 mL) was added
to the flask and the solution was stirred for 15 min; the
deltamethrin trifluoroacetate intermediates were not isolated.
The reaction mixture was extracted three times into ethyl
acetate and then washed with water and brine (saturated
NaCl), and the combined organic layer was concentrated. The
residue was chromatographed on silica four times [first with
n-hexane/ethyl acetate (1:1) and three times with benzene/

Hapten Design for Pyrethroid Immunoassays
J. Agric. Food Chem., Vol. 46, No. 2, 1998 523
(S)-R-Cyano-[3′-(4′′-(2-carboxyethylcarbonyloxy)phenoxy)-
phenyl]methyl (1R,3R)-3-(2′,2′-Dibromoethenyl)-2,2-dimethyl-
cyclopropanecarboxy Succinimidyl Ester. The acid 4 (42.4 mg,
0.068 mmol) was treated as for the acid 3 to give a pale yellow
oil. The reaction scheme and structures of the compounds are
shown in Skerritt and Lee (1996).
120.9 (C2), 120.4, 120.3, 120.2 (C2′, C4′), 119.01, 118.9 (C4),
90.9 (dCBr2), 75.0 (CHCOO), 37.5, 37.2, 37.1 (CH3), 32.7, 32.5,
32.4 (CH3), 29.6, 29.57, 29.53 [C(CH3)], 29.4, 29.3, 29.2 (CHCO)
and 16.2, 16.1 (CHC).
(()-1-Carboxy-(3′-phenoxyphenyl)methyl 3-(2′,2′-Dichloro-
ethenyl)-2,2-dimethylcyclopropanecarboxylate (9). The acid 6
(0.47 g, 2.6 mmol) was treated with 10 mL of SOCl2 (caution)
to yield the acid chloride as a light brown oil. 3-Phenoxyman-
delic acid (633 mg, 2.6 mmol) was added to a flask containing
the acid chloride prepared from 6 (2.6 mmol), and the mixture
was stirred for 3 days. It was extracted and concentrated in
the same manner as for compound 8, and the residue was
chromatographed on silica [ethyl acetate/petroleum ether/
acetic acid (20:79.9:0.1)] to yield a light brown oil (780 mg,
70%): TLC Rf 0.2 [ethyl acetate/petroleum ether/acetic acid
(3:6.9:0.1)]; 1H NMR (CDCl3) δ 9.55 (bs, COOH), 7-7.4 (m,
aromatic H), 6.24 (d, dCH, J ) 8.8 Hz), 6.24 (d, dCH, J ) 8.6
Hz), 5.89, 5.92 (2 × s, CHCOO), 2.01 (m, CHC, CHCO), and
1.25, 1.27, 1.28, 1.30 (4 × s, 2 × CH3); 13C NMR δ 174.5, 174.3
(COOH), 170.6, 170.5 (CO), 158.4, 157.3 (C3, C1′), 135.9, 135.7
(C1), 130.9 (C5), 130.6 (C3′, C5′), 125.2, 125.0 (C6), 124.4, 124.3
(dCH), 122.9, 122.9 (C4′), 121.9, 121.8 (C2), 119.9, 119.8 (C2′,
C6′), 118.7, 118.6 (C4), 118.2 (dCCl2), 103.6 (dCCl2), 75.6, 74.5
(CHCOO), 33.9, 33.8 (CH3), 32.1, 31.9 (CH3), 29.2 [C(CH3)],
29.0, 28.9 (CHCO), and 15.7, 15.6 (CHC).
(()-1-Carboxy-(3′-phenoxypheny)methyl 3-(2′-Chloro-3′,3′,3′-
trifluoropropenyl)-2,2-dimethylcyclopropanecarboxylate (10).
The acid 7 (1 g, 4.66 mmol) was also treated with 10 mL of
SOCl2 (caution) to yield the acid chloride as a light brown oil.
The acid chloride (4.7 mmol) was treated with 3-phenoxyman-
delic acid (379 mg, 1.55 mmol) to yield a light brown oil (610
mg, 86%): 1H NMR (CDCl3) δ 7-7.4 (m, aromatic H), 6.89 (d,
dCH, J ) 9.2), 6.86 (d, dCH, J ) 9.7), 6.82 (d, dCH, J ) 9.2),
6.81 (d, dCH, J ) 9.1), 5.87, 5.88 (2 × s, CHCOO), 2.2 (m,
CHC), 2.10 (d, CHCO, J ) 8.4), 2.09 (d, CHCO, J ) 5.9), and
1.27, 1.28, 1.30, 1.33 (4 × s, 2 × CH3); 13C NMR δ 191.6
(COOH), 170.5, 170.3 (CO), 137.7, 135.2 (C3, C1′), 132.1, (C1),
130.4 (C5), 130.3 (C3′, C5′), 129.5 (dCH), 124.8, 124.7 (dCH,
C6), 123.1 (C4′), 121.3, 121.8, 122.1, 122.2 (CF3), 120.5 (C2),
120.3 (C2′, C6′), 119.1 (C4), 118.6, 118.1 (dCCF3), 60.6, 60.8
(CHCO), 33.0 (CH3), 30.7 (CH3), 28.5 [C(CH3)], 28.4 (CHCO),
and 14.9 (CHC).
Con ju ga tion th r ou gh th e Mid d le of th e Molecu le
(F igu r e 1). 1. Hydrolysis of Pyrethroids. 3-(2′,2′-Dibromo-
ethenyl)-2,2-dimethylcyclopropanecarboxylic Acid (5). To a
flask containing deltamethrin (15.4 g, 0.31 mol) was added 300
mL of 1 M NaOH in 60% tetrahydrofuran (THF), and the
mixture was refluxed for 3 days. The resulting solution was
acidified using concentrated HCl (caution) and extracted three
times with ethyl acetate. The combined organic layers were
then washed (0.1 M HCl, water, and brine), dried over MgSO4,
and concentrated under reduced pressure. The residue was
chromatographed on silica using flash chromatography [ethyl
acetate/petroleum ether/acetic acid (10:89.5:0.5)] to give 5 as
a white solid, which was recrystallized to give white needle-
like crystals (5.0 g, 55%): mp 108-110 °C; TLC Rf 0.43 [ethyl
acetate/petroleum ether/acetic acid (20:79.9:0.1)]; 1H NMR
(CDCl3) δ 6.72 (d, dCH, J ) 8.6 Hz), 2.04 (t, CHC, J ) 8.5
Hz), 1.88 (d, CHCO, J ) 8.5 Hz), 1.29 (s, CH3), and 1.28 (s,
CH3); 13C NMR δ 171.1 (COOH), 133.0 (dCH), 89.8 (dCBr2),
36.4, 31.6 (2 × CH3), 28.5 [C(CH3)], 28.5 (CHCO), and 15.0
(CHC).
3-(2′,2′-Dichloroethenyl)-2,2-dimethylcyclopropanecarbox-
ylic Acid (6). To R-cypermethrin (10 g, 24 mmol) was added
300 mL of 1 M NaOH in 60% THF, and the mixture was
refluxed for 36 h. The mixture was extracted in the same
manner as for deltamethrin, and the residue was chromato-
graphed on silica [ethyl acetate/petroleum ether/acetic acid (2:
7.9:0.1)] to yield 6 as a white solid. After recrystallization from
ethyl acetate/petroleum ether, white crystals were obtained
(3.12 g, 73.4%): mp 89-91 °C; TLC Rf 0.45 [ethyl acetate/
petroleum ether/acetic acid (2:7.9:0.1)]; 1H NMR (CDCl3) δ 6.18
(d, dCH, J ) 8.7 Hz), 2.09 (t, CHC, J ) 8.5 Hz), 1.84 (d, CHCO,
J ) 8.6 Hz), 1.26 (s, CH3), and 1.25 (s, CH3); 13C NMR δ 177.4
(COOH), 124.4 (dCH), 121.1 (dCCl2), 33.4, 31.7 (2 × CH3),
28.5 (CHCO), 28.5 [C(CH3)], and 14.9 (CHC).
3-(2′-Chloro-3′,3′,3′-trifluoropropenyl)-2,2-dimethylcyclopro-
panecarboxylic Acid (7). λ-Cyhalothrin (10.3 g, 22.9 mmol) was
treated in the same manner as for R-cypermethrin, and after
recrystallization from ethyl acetate/petroleum ether yielded
crystals (2.35 g, 47.9%): mp 115-117 °C; TLC Rf 0.38 [ethyl
acetate/petroleum ether/acetic acid (2:7.9:0.1)]; 1H NMR (CDCl3)
δ 6.84 (d, dCH, J ) 9.3 Hz), 2.04 (t, CHC, J ) 9.2 Hz), 1.88
(d, CHCO, J ) 8.4 Hz), 1.30 (s, CH3), and 1.29 (s, CH3); 13C
NMR δ 177.1 (COOH), 129.6, 129.5, 129.49, 129.43 (dCH),
122.4, 122.2, 121.9 (CF3), 118.6 (dCCF3), 32.6 (CH3), 31.6
(CH3), 29.6 (C(CH3)), 28.4 (CHCO), and 14.8 (CHC).
2. Generation of Esters via Acid Chlorides. (()-1-Carboxy-
(3′-phenoxyphenyl)methyl 3-(2′,2′-Dibromoethenyl)-2,2-meth-
ylcyclopropanecarboxylate (8). The acid 5 (0.86 g, 3.0 mmol)
was treated with thionyl chloride (SOCl2, 5 mL, caution) by
stirring under reflux for 1 h. The solution was concentrated
under reduced pressure to remove excess SOCl2, and the
resulting yellow liquid was purified by bulb-to-bulb distillation.
3-Phenoxymandelic acid was prepared by treating 3-phenoxy-
benzaldehyde cyanohydrin (Aldrich) with concentrated HCl
(caution). 3-Phenoxymandelic acid (104 mg, 0.43 mmol) was
added to the flask containing 1 mL of acid chloride 8 and
stirred for 6 days, after which time 1,4-dioxane (1 mL) and
saturated aqueous NaHCO3 (2 mL) were added and stirred
for 1 h. The solution was acidified with concentrated HCl
(caution) to pH 1 and extracted three times with ethyl acetate.
The combined organic layers were washed with water and
brine, dried over MgSO4, and concentrated. The residue was
chromatographed on silica [chloroform/acetic acid (99.5:0.5)]
to give a white solid (109 mg, 49%): 1H NMR (CDCl3) δ 9.6
(bs, COOH), 7-7.4 (m, aromatic H), 6.72 (d, dCH, J ) 8.6
Hz), 5.86, 5.89 (2 × s, CHCOO), 2.0 (m, CHC), 1.8 (d, CHCO,
J ) 8.4 Hz), and 1.27 (m, 2 × CH3); 13C NMR δ 178.7, 178.1
(COOH), 159 (C3, C1′), 134.1, 134, 133.9 (C1), 131.3, 131.0,
130.9 (C5), 125.9 (dCH), 125.2, 12.9, 12.8 (C6), 123.3 (C4′),
3. Preparation of Carbamoyl Derivatives. N-2-(1′,1′-Di-
methylethyloxycarbonylethyl)-carbamoyl-(3′-phenoxyphenyl)-
methyl 3-(2′,2′-Dibromoethenyl)-2,2-dimethylcyclopropanecar-
boxylate (11). The flask containing the acid 8 (46 mg, 0.09
mmol) in 2 mL of dichloromethane was cooled in an ice bath
for 30 min, and then DCC (23 mg, 0.1 mmol) followed by
DMAP (0.5 mg) was added and stirred for 30 min. Finally,
tert-butyl 3-aminopropanoate (26 mg, 0.1 mmol) was added,
and the mixture was stirred overnight. After filtration of the
solution to remove the precipitate of dicyclohexylurea, the
mixture was extracted with ethyl acetate and the organic layer
was washed (0.1 M HCl, saturated NaHCO3, water, and brine),
dried, and concentrated. The residue was chromatographed
on silica [ethyl acetate/petroleum ether (30:70)] to give the tert-
butyl ester (21 mg, 37%): 1H NMR (CDCl3) δ 7-7.4 (m,
aromatic H), 6.22 (d, dCH, J ) 8.7), 6.18 (d, dCH, J ) 8.5
Hz), 5.98, 6.00 (2 × s, CHCOO), 3.49 (m, CH2N), 2.44 (m, CH2-
CO), 2.09 (m, CHC, CHCO), 1.44 (s, tert-butyl), and 1.25, 1.26
(2 × s, 2 × CH3).
N-2-(1′,1′-Dimethylethyloxycarbonylethyl)carbamoyl-(3′-phe-
noxyphenyl)methyl 3-(2′,2′-Chloroethenyl)-2,2-dimethylcyclo-
propanecarboxylate (12). The acid 9 (260 mg, 0.6 mmol) was
treated in the same manner as for compound 8 to produce a
light brown oil (82 mg, 23%): 1H NMR (CDCl3) δ 7-7.4 (m,
aromatic H), 6.22 (d, dCH, J ) 8.7 Hz), 6.18 (d, dCH, J ) 8.5
Hz), 5.98, 6.0 (2 × s, CHCOO), 3.49 (m, CH2N), 2.44 (m, CH2-
CO), 2.09 (m, CHC, CHCO), 1.44 (s, tert-butyl), and 1.25, 1.26
(2 × s, 2 × CH3).
N-2-(1′,1′-Dimethylethyloxyca rbonylethyl)ca rba moyl-(3′-
phenoxyphenyl)methyl 3-(2′-Chloro-3′,3′,3′-trifluoropropenyl)-
2,2-dimethylcyclopropanecarboxylate (13). The acid 10 (242
mg,0.55 mmol) was also treated in the same manner as for
compound 8 to produce a light brown oil (265 mg, 87%): 1H

524 J. Agric. Food Chem., Vol. 46, No. 2, 1998
Lee et al.
NMR (CDCl3) δ 7-7.4 (m, aromatic H), 6.82 (d, dCH, J ) 9.6
Hz), 5.94, 5.97 (2 × s, CHCOO), 3.46 (m, CH2N), 2.4 (m, CH2-
CO), 2.10 (d, CHCO, J ) 9.0 Hz), 1.40, 1.43 (2 × s, tert-butyl),
and 1.23, 1.27, 1.28 (3 × s, 2 × CH3).
without further purification since NMR showed complete
oxidation of the double bond: TLC Rf 0.6 [ethyl acetate/
petroleum ether/acetic acid (30.69.9:0.1)] and Rf 0.38 [chloroform/
petroleum ether/acetic acid (90:9.9:0.1)]; 1H NMR (CDCl3) δ
3.47 (m, CH2N), 2.49 (m, CH2CO), 2.08 (d, CHC, J ) 7.3 Hz),
1.95 (t, CHCO, J ) 8.8 Hz), 1.41, 1.42, 1.44, 1.45 (4 × s, tert-
butyl), and 1.26, 1.27 (2 × s, CH3).
4. Removal of tert-Butyl Protecting Group. N-2-(Carboxy-
ethenyl)carbamoyl-(3′-phenoxyphenyl)methyl 3-(2′,2′-Dibromo-
ethenyl)-2,2-dimethylcyclopropanecarboxylate (14) and Suc-
cinimidyl Ester. TFA (1 mL, caution) was added to the ester
11 (21 mg, 0.03 mmol), and the mixture was stirred for 10
min. The excess TFA was removed under reduced pressure
to yield an acid 14, which was used in the next step without
further purification. The 1H NMR spectrum for 14 showed
the disappearance of the tert-butyl peak at 1.4 ppm. DCC (8
mg, 0.04 mg) and DMAP (0.2 mg) were added to the acid in
dichloromethane (2 mL), which was cooled in an ice bath and
stirred for 30 min, and then NHS (7 mg, 0.035 mmol) was
added, and the mixture was stirred overnight. The dicyclo-
hexylurea precipitate was removed by filtration, and the
filtrate was concentrated to yield a viscous yellow oil (16 mg,
80%).
(()-Cyano-(3-phenoxyphenyl)methyl N-2-(1′,1′-Dimethyleth-
yloxycarbonylethyl)carbamoyl-2,2-dimethylcyclopropanecar-
boxylate (19). To the monoester 18 (298 mg, 1.05 mmol) in
dichloromethane cooled in ice bath was added DCC (286 mg,
1.38 mmol), and the mixture was stirred for 15 min. Phe-
noxybenzylcyanohydrin (311 mg, 1.38 mmol, caution) was then
added to the mixture, and stirring continued overnight. After
the solution was filtered and concentrated, the residue was
dissolved in ethyl acetate and washed with water and brine.
The ester was obtained as a yellow oil (167 mg, 28.7%) after
chromatography on silica [ethyl acetate/petroleum ether (25:
75)]; 1H NMR (CDCl3) δ 7-7.4 (m, aromatic H), 6.31 (s,
CHCN), 3.43 (m, CH2N), 2.41 (m, CH2CO), 2.08 (d, CHC, J )
7.3 Hz), 1.42 (s, tert-butyl), and 1.15, 1.16 (2 × s, 2 × CH3).
N-2-(Ca rboxyethyl)ca rba moyl-(3′-phenoxyphenyl)methyl
3-(2′,2′-Chloroethenyl)-2,2-dimethylcyclopropanecarboxylate (15)
and Succinimidyl Ester. The tert-butyl ester 12 (80.7 mg, 0.16
mmol) was also treated with 1 mL of TFA (caution) to yield
acid 15, and it was treated with NHS (21.5 mg, 0.19 mmol),
DCC (39.4 mg, 0.19 mmol), and DMAP (0.98 mg) to yield a
light yellow oil (79.3 mg, 89%): 1H NMR for 15 (CDCl3) δ 7-7.4
(m, aromatic H), 6.21 (d, dCH, J ) 8.8 Hz), 6.18 (d, dCH, J )
8.7 Hz), 5.99, 6.02 (2 × s, CHCOO), 4.15 (dd, CH2N, J ) 8.5
and 6.9 Hz), 3.15 (m, CH2CO), 2.13 (d, CHCO, J ) 8.6 Hz),
1.99 (d, CHCO, J ) 8.4 Hz), and 1.26, 1.29 (2 × s, 2 × CH3).
N-2-(Carboxyethyl)carbamoyl-(3′-phenoxyphenyl)methyl 3-
(2′-Ch lor o-3′,3′,3′-tr iflu or opr open yl)-2,2-d im eth ylcyclo-
propanecarboxylate (16) and Succinimidyl Ester. The tert-
butyl ester 13 (242 mg, 0.55 mmol) was also treated in the
same manner as for 11 to yield 16: the 1H NMR spectrum
showed the disappearance of tert-butyl peaks at 1.4 ppm. Then
the acid was treated with DCC, DMAP, and NHS to produce
an active ester as a light brown oil (265 mg, 79%).
(()-Cyano-(3-phenoxyphenyl)methyl N-2-(Carboxyethyl)car-
bamoyl-2,2-dimethylcyclopropanecarboxylate (20) and Succin-
imidyl Ester. The monoester was treated with TFA for 15 min
(caution) to produce an acid, 20. The cleavage of the tert-butyl
group was confirmed by NMR: 1H NMR (CDCl3) δ 8.9 (bs,
COOH), 7.0-7.4 (m, aromatic H), 6.31 (s, CHCN), 3.83 (m,
CH2N), 2.66 (m, CH2CO), and 1.26 (m, 2 × CH3); 13C NMR δ
176.4 (COOH), 174.8 (CO), 169.5, 168.3 (COCN), 158.0, 156.0
(C3, C1′), 133.2 (C1), 130.6 (C5), 129.9 (C3′, C5′), 124.1, 124.0,
123.9 (C6, dCH), 122.2 (C4′), 120.2 (C2), 119.4 (C2′, C6′), 117.8
(C4), 35.6, 35.1 (CH2CN), 33.6, 33.5 (CHCO), 31.9, 31.4 (CH2-
CO), 26.2, 25.4 (CHCO), 29.7 [C(CH3)], 20.4 (CH3), 19.9 (CH3).
The active ester was obtained by adding NHS (65 mg, 027
mmol) to a solution containing the acid 20 (62 mg, 0.23 mmol),
DCC (57 mg, 0.27 mmol), and DMAP (5 mg) in dichlo-
romethane (10 mL) and stirred overnight, before filtration and
concentration to yield a yellow oil (80%).
Cou p lin g On ly t h e Cyclop r op a n e Moiet y. N-2-(1′,1′-
Dimethylethyloxycarbonylethyl) 2,2-Dimethyl-3-(2′,2′-bromo-
ethenyl)cyclopropanecarboxyamide (21). The acid 5 (200 mg,
0.67 mmol) in dichloromethane was cooled in ice bath for 15
min. DCC (94 mg, 0.81 mmol) followed by DMAP (4 mg) was
added to the solution and stirred for a further 30 min in the
ice bath. tert-Butyl 3-aminopropanoate (117 mg, 0.81 mmol)
was then added to the solution and stirred overnight. The
precipitates of dicyclohexylurea were removed by filtration
through Celite, and the filtrate was extracted in diethyl ether
twice and concentrated to a pale brown oil, which was used
without further purification: 1H NMR (CDCl3) δ 6.75 (d, dCH,
J ) 8.6 Hz), 4.07 (t, CH2N, J ) 6.4 Hz), 3.67 (t, CH2CO, J )
6.4 Hz), 2.34 (m, CHC), 1.86 (d, CHCO, J ) 7.3 Hz), 1.43 (s,
tert-butyl), and 1.23, 1.25 (2 × s, 2 × CH3).
Cou p lin g a t t h e Cyclop r op a n e Moiet y (F igu r e 2).
Preparation of Chrysanthemic Acid. To chrysanthemum mono-
carboxylic acid ethyl ester (4.52 g, 23 mmol; 2,2-dimethyl-3-
isobutenylcyclopropane-1-carboxylic acid, Sigma) in 80% eth-
anol was added KOH (2.4 g, 42.8 mmol), and the mixture was
refluxed at 80 °C overnight. The acidified solution (pH 2) was
extracted twice with ethyl acetate, and the combined organic
layers were washed with 0.1 M HCl, water, and brine, dried
over MgSO4, then concentrated to give a white solid (3.82 g,
99%); TLC Rf 0.39 [ethyl acetate/petroleum ether (3:7)].
N-2-(1′,1′-Dimethylethyloxycarbonylethyl) 2,2-Dimethyl-3-(2′-
methylpropenyl)cyclopropanecarboxamine (17). To chrysan-
themum monocarboxylic acid (500 mg, 2.63 mmol) in dichlo-
romethane (20 mL) cooled in an ice bath for 15 min were added
DCC (308 mg, 2.63 mmol) and DMAP (20 mg), and the mixture
was stirred for a further 15 min. tert-Butyl 3-aminopropanoate
(382 mg, 2.63 mmol) was then added, and the mixture was
stirred overnight. After filtration and concentration, the
residue was chromatographed on silica [chloroform/acetic acid
(99.9:0.1)] to yield a light yellow oil (381.7 mg, 49%): TLC Rf
0.6 [chloroform/acetic acid (99.9:0.1)] and Rf 0.7 [ethyl acetate/
N-2-(1′,1′-Dimethylethyloxycarbonylethyl) 2,2-Dimethyl-3-
(2′,2′-dichloroetheyl)cyclopropanecarboxyamide (22). The acid
6 (200 mg, 0.96 mmol) was treated in the same manner with
DCC (135 mg, 1.15 mmol), DMAP (7 mg), and tert-butyl
3-aminopropanoate (167 mg, 1.15 mmol) to produce a pale
yellow oil, which was used without further purification, as the
TLC spots were only weakly UV active: 1H NMR (CDCl3) δ
6.92 (d, dCH, J ) 9.4 Hz), 6.91 (d, dCH, J ) 9.5 Hz), 4.1 (t,
CH2N, J ) 6.4 Hz), 3.67 (t, CH2CO, J ) 6.4 Hz), 2.16 (t, CHC,
J ) 8.9 Hz), 2.00, (m, CHC), 1.86 (d, CHHCO, J ) 8.9 Hz),
1.43 (s, tert-butyl), 1.30, 1.31 (2 × s, CH3), and 1.30 (s, 3 H).
1
petroleum ether/acetic acid (30:69.9:0.1)]; H NMR (CDCl3) δ
6.17 (bs, NH), 5.36 (d, 1 H, J ) 8.61 Hz), 4.9 (d, dCH, J )
7.74 Hz), 4.9 (d, dCH, J ) 7.9 Hz), 3.4 (m, CH2N), 2.4 (t, CH2-
CO, J ) 6.03 Hz), 2.08 (d, CHCO, J ) 7.3 Hz), 1.95 (t, CHC,
J ) 8.8 Hz), 1.65, 1.73 [2 × s, ((CH3)2Cd] 1.4 (s, tert-butyl),
and 1.05, 1.13, 1.15, 1.17 (4 × s, 2 × CH3).
N-2-(1′,1′-Dimethylethyloxycarbonylethyl) 2,2-Dimethyl-3-(2′-
chloro-3′,3′,3′-trifluoropropenyl)cyclopropanecarboxyamide (23).
The tert-butyl ester of 3-(2-chloro-3,3,3-trifluoropropenyl)-2,2-
dimethylcyclopropanecarboxylic acid (200 mg, 0.93 mmol) was
prepared in a similar manner to yield an amber oil. This was
used in the next step without futher purification, due to lack
of sufficient UV activity: 1H NMR (CDCl3) δ 6.93 (d, dCH, J
) 9.4 Hz), 5.7 (bs, NH), 4.1 (t, CH2N, J ) 6.5 Hz), 2.31 (t,
CH2CO, J ) 7.5 Hz), 2.00, (m, CHC), 1.86 (d, CHCO, J ) 8.9
Hz), 1.43 (s, tert-butyl), and 1.30, 1.31 (2 × s, CH3).
N-2-(1′,1′-Dimethylethyloxycarbonylethyl)carbamoyl 2,2-
Dimethylcyclopropanecarboxylate (18). The ester 17 (406 mg,
1.38 mmol) in tert-butyl alcohol (7 mL) was added to a mixture
containing 25 mL of 0.22 M NaIO4, 3.64 mM KMnO4, and 84
mM Na2CO3 and stirred overnight. The solution was acidified
to pH 2 and extracted twice with ethyl acetate. The combined
organic layer was washed with 0.1 M Na2S2O5, water, and
brine and then dried and concentrated to give a light brown
oil (298 mg, 76%). The crude oil was used in the next step

Hapten Design for Pyrethroid Immunoassays
J. Agric. Food Chem., Vol. 46, No. 2, 1998 525
N-2-(Carboxyethyl) 2,2-Dimethyl-3-(2′,2′-dibromoetheyl)cy-
clopropanecarboxyamide (24), N-2-(Carboxyethyl) 2,2-Di-
methyl-3-(2′,2′-dichloroetheyl)cyclopropanecarboxyamide (25),
and N-2-(Carboxyethyl) 2,2-Dimethyl-3-(2′-chloro-3′,3′,3′-tri-
fluoropropenyl)cyclopropanecarboxyamide (26). The com-
pounds 21-23 were treated with TFA (1 mL, caution) to yield
an acid, and the excess TFA was removed under reduced
pressure. The clevage of the tert-butyl group was confirmed
by proton NMR, and the crude acids were used in the next
(3:7)]; 1H NMR (CDCl3) δ 7-7.4 (m, aromatic H), 4.2 (m, 7
lines, OCH2), 2.65 (m, 14 lines, A2B2), 0.98 (m, CH2Si), 0.023
[s, Si(CH3)3].
(()-Cyano-(3′-phenoxyphenyl)methyl 2-Carboxyethylcarbox-
ylate (28) and Succimidyl Ester. To 27 in acetonitrile (30 mL)
was added tetraethylammonium fluoride (TEAF; 671 mg, 4.5
mmol), and the mixture was refluxed at 90 °C for 1 h. The
solution was concentrated, and the residue was dissolved in
ethyl acetate. The organic layer was washed with water and
brine, dried (over MgSO4), and concentrated to yield a bright
yellow product (42 mg): TLC Rf 0.46 [ethyl acetate/petroleum
ether (3:7)]. The acid (42 mg, 0.13 mmol) was treated with
DCC (34 mg, 0.16 mmol), DMAP (0.8 mg), and NHS (19 mg,
0.16 mmol) in dichloromethane (5 mL) at 0 °C for 6 h. The
byproduct dicyclohexylurea was removed by filtration through
Celite and the filtrate concentrated.
Ha p t en s Ut ilizin g Bu lk y Sp a cer s. N-4′-(2′-Trimethyl-
silylethyloxycarbonyl)cyclohexylmethylcarbamoylmethyl 3-(2′,2′-
Dibromoethenyl)-2,2-dimethylcyclopropanecarboxylate (29). 2-
(Trimethylsilyl)ethyl trans-4-aminomethylcyclohexanecarb-
oxylate (250 mg, 1 mmol; Hill et al., 1993) was added to a flask
containing the acid 5 (250 mg, 0.84 mmol) and DCC (207 mg,
1 mmol) in dichloromethane cooled in an ice bath and stirred
overnight. The mixture was filtered through Celite to remove
dicyclohexylurea, and the filtrate was partitioned between
dichloromethane and water/brine and then dried over MgSO4.
Following the concentration, the residue was chromatographed
on silica [ethyl acetate/petroleum ether (2:8)] to obtain an
alkene, 29 (352 mg, 78.5%): 1H NMR (CDCl3) δ 6.94 (d, dCH,
J ) 8.8 Hz), 5.9 (bs, NH), 4.15 (m, OCH2), 3.17 (t, NCH2, J )
8.0), 1.0-2.3 (m, cyclohexyl), 1.99 (t, CHC, J ) 7.9 Hz), 1.81
(d, CHCO, J ) 8.5 Hz), 1.23, 1.24 (2 × s, CH3), 0.92 (m, CH2-
Si), and 0.02 [s, Si(CH3)3].
N-(4′-Cyclohexylmethyl)carbamoylmethyl 3-(2′,2′-Dibromo-
ethenyl)-2,2-dimethylcyclopropane carboxylate (30) and Suc-
cinimidyl Ester. To the alkene 29 (352 mg, 0.785 mmol) in
acetonitrile was added TEAF (351.5 mg, 2.36 mmol), and the
solution was refluxed for 2 h. The residue was dissolved in
dichloromethane after concentration and extracted with 1 M
HCl, water, and brine, and then dried (MgSO4). The residue
was chromatographed on silica [ethyl acetate/petroleum ether
(1:1)] to yield an acid 30. The acid (300 mg, 0.79 mmol) was
then treated with DCC (178.6 mg, 0.86 mmol), DMAP (10 mg),
and NHS (90.4 mg, 0.79 mmol) in dichloromethane to produce
a succinimidyl ester; 1H NMR for 30 (CDCl3) δ 6.89 (d, dCH,
J ) 8.8 HZ), 5.69 (bs, NH), 3.09 (t, NCH2, J ) 8.0), 1.0-2.3
(m, cyclohexyl), and 1.15, 1.26 (2 × s, 2 × CH3).
1
step without further purification. The H NMR spectral data
of 24-26 were as follows: (24) (CDCl3) δ 6.76 (d, dCH, J )
8.6 Hz), 4.10 (t, CH2N, J ) 6.3 Hz), 2.45 (t, CH2CO, J ) 7.4
Hz), 1.93 (t, CHC, J ) 8.5 Hz), 1.82 (d, CHCO, J ) 8.4 Hz),
and 1.23, 1.24, 1.25, 1.27 (4 × s, CH3); (25) (CDCl3) δ 6.90 (d,
dCH, J ) 9.5 Hz), 6.89 (d, dCH, J ) 9.4 Hz), 4.35 (t, CH2N,
J ) 6.1 Hz), 2.47 (t, CH2CO, J ) 7.3 Hz), 2.16 (t, CHC, J )
7.4 Hz), 1.96 (d, CHCO, J ) 8.9 Hz), and 1.28, 1.29 (2 × s,
CH3); (26) (CDCl3) δ 6.89 (d, dCH, J ) 11 Hz), 6.88 (d, dCH,
J ) 10.4 Hz), 4.12 (t, CH2N, J ) 6.3 Hz), 2.45 (t, CH2CO, J )
7.3 Hz), 2.21 (t, CHC, J ) 7.8 Hz), 1.97 (d, CHCO, J ) 8.3
Hz), 1.97 (d, CHCO, J ) 8.4 Hz), and 1.27, 1.28, 1.3 (3 × s,
CH3).
N-2-(Carboxyethyl) 2,2-Dimethyl-3-(2′,2′-dibromoetheyl)cy-
clopropanecarboxyamide Succinimidyl Ester. To a solution
containing acid 21 (108.4 mg, 0.31 mmol) and dichloromethane
(5 mL) cooled in an ice bath were added DCC (75.8 mg, 0.37
mmol) and DMAP (20 mg) and stirred for 30 min; NHS (42.2
mg, 0.37 mmol) was then added, and the mixture was stirred
overnight. The dicyclohexylurea precipitate was removed by
filtration, and the filtrate was concentrated to yield an off-
white solid: 1H NMR (CDCl3) δ 6.78 (d, dCH, J ) 8.5 Hz),
6.62 (d, dCH, J ) 8.1 Hz), 4.16 (t, CH2N, J ) 6.2 Hz), 2.85
(bs, 2 × CH2CON), 2.75 (t, CH2CO, J ) 7.2 Hz), 2.20 (t, CHC,
J ) 8.3 Hz), 2.11 (t, CHC, J ) 8.3 Hz), 1.97 (t, CHC, J ) 8.5
Hz), 1.88 (d, CHCO, J ) 8.4 Hz), 1.26, 1.27 (2 × s, CH3); 13C
NMR δ 171.9, 169.2, 168.2 (4 × CON), 89.4 (dCBr2), 37.1
(CH2N), 35.8 (CH2CO), 33.8 (CH3), 31.8 (CH3), 27.9 (CHCO),
25.7 (2 × CH2CON), and 15.2 (CHC).
N-2-(Carboxyethyl) 2,2-Dimethyl-3-(2′,2′-dichloroetheyl)cy-
clopropanecarboxyamide Succinimidyl Ester and N-2-(Car-
boxyethyl) 2,2-Dimethyl-3-(2′-chloro-3′,3′,3′-trifluoropropenyl-
cyclopropanecarboxyamide Succinimidyl Ester. The acids 22
and 23 were also treated with DCC, DMAP, and NHS in a
normal manner to produce white solids. The 1H NMR spectral
data for succinimidyl ester 22 were as follows: δ 6.92 (d, dCH,
J ) 9.0 Hz), 4.20 (t, CH2N, J ) 6.0 Hz), 3.73 (m, CH2CO),
2.87 (bs, 2 × CH2CON), 2.13 (t, CHC, J ) 6.0 Hz), 2.05 (d,
CHCO, J ) 8.4 Hz), and 1.25, 1.26, 1.27, 1.28, 1.32, 1.34 (6 ×
s, 2 × CH3); 13C NMR δ 170.0, 169.0, 168.0 (4 × CON), 130.0,
129.9 (dCCl2), 32.6 (CH2N), 30.9 (CH2CO), 29.3 (CHCO), 25.5
(2 × CH2CON), and 14.8 (CHC). The 1H NMR spectral data
for the succinimidyl ester of 23 were as follows: δ 6.91 (d,
dCH, J ) 9.3 Hz), 6.73 (d, CH, J ) 9.3 Hz), 6.72 (d, dCH, J
) 9.0 Hz), 4.11 (t, CH2N, J ) 6.5 Hz), 2.83 (bs, 2 × CH2CON),
2.45 (t, CH2CO, J ) 7.4 Hz), 2.39 (t, CH2CO, J ) 9.0 Hz), 2.16
(t, CHC, J ) 9.3 Hz), 2.15 (t, CHC, J ) 8.6 Hz), 1.97 (d, CHCO,
J ) 8.3 Hz), and 1.27, 1.28 (2 × s, CH3); 13C NMR δ 172.4,
170.8, 169.4, 166 (4 × CON), 130.3, 130.2, 128.3, 128.2 (dCH),
122.4, 121.9 (CF3), 118.6, 188.4, (dCCl), 33.8 (CH2N), 32.3
(CH3), 31.1 (CH3), 30.5 (CH2CO), 28.6 (CHCO), 15.1, 15.0
(CHC).
Cou p lin g On ly th e P h en oxyben zyl Moiety. (()-Cyano-
(3′-phenoxyphenyl)methyl 2-Trimethylsilylethyloxycarbonyl-
ethylcarboxylate (27). (()-3-Phenoxybenzylaldehyde cyano-
hydrin (1 mL, 3.1 mmol) and 2-trimethylsilylethyl oxycarbo-
nylethylcarboxylic acid (762 mg, 3.5 mmol) in dichloromethane
(30 mL) were cooled to 0 °C in an ice bath for 15 min. DCC
(767 mg, 3.7 mmol) was added followed by DMAP (20 mg),
and the mixture was stirred at room temperature for 6 h. After
dicyclohexylurea was removed by filtration, the filtrate was
extracted with ethyl acetate, water (slightly acidified), and
brine. The organic layer was dried over MgSO4 and concen-
trated to produce a red oil (unable to determine the percentage
yield as product was suspected to be contaminated with
dicyclohexyl urea): TLC Rf 0.57 [ethyl acetate/petroleum ether
N -4′-(2′-T r im et h ylsilylet h yloxyca r bon yl)cycloh exyl-
methylcarbamoylmethyl 3-Phenoxybenzamide (31). To 3-phen-
oxybenzoic acid (500 mg, 2.3 mmol) and DCC (496.8 mg, 2.4
mmol) in 10 mL of dichloromethane was added 2-(trimethyl-
silyl)ethyl trans-4-aminomethylcyclohexanecarboxylate (600
mg, 2.4 mmol), and the mixture was stirred overnight in an
ice bath. After the solution was extracted with water and
brine and concentration following the filtration, the residue
was chromatographed on silica [methanol/chloroform (1:99)]
to produce 31 (206.3 mg, 20%): 1H NMR (CDCl3) δ 7-7.45
(m, aromatic H), 6.44 (bt, NH, J ) 5.3 Hz), 4.42 (t, OCH2, J )
8.3), 3.28 (t, NCH2, J ) 6.5 Hz), 1.0-2.3 (m, cyclohexyl), 0.98
(m, CH2Si), and 0.02 [s, Si(CH3)3]; 13C NMR δ 175.9 (COOC),
166.9 (CON), 157.4, 156.3 (C3, C1′), 136.3 (C1), 129.7 (C3′, C5′),
123.5 (C6), 121.3 (C6′), 121.2 (C2), 118.9 (C2′, C4′), 117.5 (C4),
62.3 (CH2O), 45.7 (CH2N), 43.2 (CHCH2N), 37.2 (CHCO2,
cyclohexyl), 32.3 (CHCO2), 29.6 (2 × CH2CHCO), 28.2 (2 ×
CH2CHCH2), 17.0 (CH2Si), and -1.7 [Si(CH3)3].
N-(4′-Cyclohexylmethyl)carbamoylmethyl 3-Phenoxybenz-
amide (32) and Succinimidyl Ester. A mixture of compound
31 (206 mg, 0.46 mmol) and TEAF (149 mg, 1 mmol) in 10
mL of acetonitrile was reacted under reflux for 2 h. The
solution was concentrated, dissolved in dichloromethane, and
extracted with 1 M HCl, water, and brine. After drying, the
residue was chromatographed on silica [ethyl acetate/petro-
leum ether/acetic acid (30:69.5:0.5)] to yield an acid 32 (100
mg, 100%). The acid (100 mg, 0.46 mmol) was then treated
with DCC (142.7 mg, 0.69 mmol), DMAP (20 mg), and NHS

526 J. Agric. Food Chem., Vol. 46, No. 2, 1998
Lee et al.
(79.4 mg, 0.69 mmol) in dichloromethane to yield a succinim-
idyl ester: 1H NMR for 32 (CDCl3) δ 7-7.4 (m, aromatic H),
6.3 (bt, NH, J ) 5.7 Hz), 3.31 (t, NCH2, J ) 6.3 Hz), 1-2.3 (m,
cyclohexyl); 13C NMR δ 180.7 (COOH), 167.4 (CON), 157.8,
156.8 (C3, C1′), 136.7 (C1), 130.1 (C3′, C5′), 123.9 (C6), 121.7
(C6′), 121.6 (C2), 119.3 (C2′, C4′), 117.5 (C4), 46.1 (CH2N), 43.2
(CHCH2N), 37.5 (CHCOOH), 30.0 (2 × CH2CHCH2N), and 28.5
(2 × CH2CHCO). 1H NMR for the succinimidyl ester of 32
(CDCl3) δ 7-7.4 (m, aromatic H), 6.3 (bs, NH), 3.38 (t, NCH2,
J ) 8.0 Hz), 2.85 (s, 2 × CH2CON), and 1-2.3 (m, cyclohexyl).
(()-3-Phenoxyphenyl-N-4′-(2′′-trimethylsilylethyloxycarbon-
yl)cyclohexylmethylcarbamoylmethyl 3-(2′,2′-Dichloroethenyl)-
2,2-dimethylcyclopropanecarboxylate (33). To a flask contain-
ing the acid 9 (200 mg, 0.46 mmol), DCC (149 mg, 0.72 mmol),
and DMAP (20 mg) was added 2-(trimethylsilyl)ethyl trans-
4-aminomethylcyclohexanecarboxylate (208.4 mg, 0.46 mmol),
and the mixture was stirred overnight in an ice bath. Fol-
lowing the filtration and concentration, the residue was
chromatographed on silica [ethyl acetate/petroleum ether (3:
7)] to obtain compound 33 (59.3 mg, 14.8%): 1H NMR (CDCl3)
δ 7-7.4 (m, aromatic H), 6.88 (d, dCH, J ) 8.0 Hz), 6.84 (d,
dCH, J ) 8.0 Hz), 5.7 (bs, NH), 4.3 (t, OCH2, J ) 6.1 Hz),
3.26 (t, NCH2, J ) 6.1 Hz), 1-2.5 (m, cyclohexyl), 1.18, 1.24
(2 × s, 2 × CH3), 0.96 (m, CH2Si), and 0.02 [s, Si(CH3)3].
(()-3-Phenoxyphenyl-N-(4′-carboxycyclohexylmethyl)carbam-
P yr eth r oid Sta n d a r d P r ep a r a tion . Pyrethroid stan-
dards at four to seven concentrations ranging from 0.1 to 1000
µg/L were prepared in 10% methanol from a 1000 mg/mL stock
solution in methanol. The standards were 10× concentrations
in methanol, diluted 1:10 with purified water. For a compari-
son, a 1000 µg/L standard was prepared in either 10%
methanol or purified water and serially diluted to appropriate
concentrations. Isomerized deltamethrin (see Results and
Discussion) was obtained by adding 5 mM NaOH to 1000 mg/
mL deltamethrin in methanol and allowed to stand at room
temperature for about 6 h. The solution was then stored at
-20 °C for use up to 4 weeks without an obvious loss of
sensitivity; however, routinely 1000 mg/L isomerized delta-
methrin in methanol was freshly prepared each week. Cyper-
methrin and λ-cyhalothrin were isomerized immediately prior
to use as these compounds were shown to hydrolyze more
rapidly than deltamethrin (see Results and Discussion). To
avoid chemical and photochemical isomerization of pyrethroids
(Ruzo et al., 1977; Maguire, 1990; Perschke and Hussain,
1992), standard curves were obtained by use of a freshly
prepared 1.0 mg/mL stock solution in methanol.
Im m u n oa ssa ys. Immunoassays were performed using an
immobilized antibody competitive immunoassay format. All
steps were performed at room temperature (18-23 °C). Mi-
crowell plates (Maxisorp, Nunc) were coated for 16 h with
antibody (100 µL/well) after dilution to 10 µg/mL in 50 mM
sodium carbonate buffer, pH 9.6. Microwells were washed
three times in 50 mM sodium phosphate/0.9% NaCl, pH 7.2
(PBS)/0.05% Tween 20 (PBS/T) and then nonspecific antibody
binding blocked with 150 µL per well of 1% BSA in PBS for 1
h. The assay was performed by the addition of 100 µL/well
diluted pyrethroid standard or sample (diluted initially in
methanol and then 1:10 in water), and 100 µL/well enzyme
conjugate (diluted in 1% BSA in PBS) and incubation for 1 h.
After the microwells were washed five times with distilled
water, color was developed by the addition of 150 µL/well
3,3′,5′-tetramethylbenzidine (Sigma)/peroxide-based substrate
(Hill et al., 1991) for 30 min. Color development was stopped
by addition of 50 µL/well 1.25 M sulfuric acid, and absorbance
values were determined at 450 nm using a microplate reader,
interfaced with a personal computer; data were fitted to a four-
parameter logistic plot using SOFTmax (Molecular Devices,
Menlo Park, CA). The optimal concentration of enzyme
conjugate was determined for each assay as the lowest
concentration required to produce assay color of 1.0-1.2 OD
units. Initial characterizations of all the combinations were
conducted by determining the percentage inhibitions for
deltamethrin, cypermethrin, and λ-cyhalothrin at 10 µg/L.
Those combinations providing >50% inhibition and utilizing
a low concentration of enzyme conjugate were selected for
further characterization.
oylmethyl
3-(2′,2′-Dichloroethenyl)-2,2-dimethylcyclopro-
panecarboxylate (34). TEAF was added to a flask containing
compound 33 (59.3 mg, 0.068 mmol) in acetonitrile (10 mL)
and was reacted under reflux for 2 h. The solution was
concentrated, and the residue was dissolved in 10 mL of
dichloromethane and extracted with 20 mL of 1 M HCl, water,
and brine. The extractant was then dried over MgSO4,
concentrated, and chromatographed on silica [ethyl acetate/
petroleum ether/acetic acid (30:69.5:0.5)] to yield acid 34 (17.4
mg, 54%). The acid was treated with DCC, DMAP, and NHS
in dichloromethane to produce a succinimidyl ester: 1H NMR
for 34: (CDCl3) δ 7-4.4 (m, aromatic H), 6.23 (d, dCH, J )
8.9 Hz), 5.0 (s, NH), 3.7 (m, CH2N), 2.1 (t, CHC, J ) 8.9 Hz),
1.88 (d, CHCO, J ) 8.4 Hz), 1.0-2.2 (m, cyclohexyl), and 1.27,
1.28, 1.29 (3 × s, 2 × CH3).
P r otein a n d En zym e Con ju ga tion . The conjugation of
NHS esters to ovalbumin (OA), keyhole limpet hemocyanin
(KLH), and horseradish peroxidase (HRP) was performed as
follows. A 20 mole excess of NHS ester was added to OA or
KLH or a 13 or 40 mole excess to HRP, with the protein being
dissolved at 5 mg/mL in 0.2 M K2HPO4, pH 9.1. The solution
was allowed to stand at 4 °C overnight, and the uncoupled
hapten was removed by desalting on a Sephadex G-25 column
(Pharmacia). Three enzyme conjugates based upon three
different haptens coupled via a spacer arm containing an
aromatic group were synthesized: a “bulky” spacer arm was
placed on haptens based on (1) cypermethrin and conjugated
through the middle of this molecule, (2) deltamethric acid, and
(3) the phenoxybenzyl moiety. The concentration of coupled
proteins was detetermined using the bicinchoninic acid assay
(Smith et al., 1985). The coupling ratio was measured by using
a method according to Plapp et al. (1971). Trinitrobenzene-
sulfonate (0.72 mg in 0.1 mL) was added to triplicate standards
containing 0, 62.5, 125, 250, 500 µg/mL and samples in 0.05
M sodium borate buffer, and the absorbance was measured
at 367 nm after 2 h.
Im m u n iza tion s. New Zealand white rabbits were im-
munized with DEL, CYP, CYH, LHDEL, LHCYP, LHCYH,
PBCY, FULPYR, and 4′OHDEL coupled to either OA or KLH,
according to the procedure described in Lee et al. (1995). The
OA and KLH conjugates of each immunogen (1 mg/mL) were
emulsified with an equal volume of Freund’s complete adju-
vant (Sigma) and injected half-subcutaneously, half-intramus-
cularly, into New Zealand White rabbits. Following two
booster injections 4 weeks apart, each containing 0.5 mg/mL
immunogen in Freund’s incomplete adjuvant (Sigma), blood
was collected from the marginal ear vein 8-10 days after the
last injection and clotted to form serum. IgG from the antisera
were purified by protein G-agarose affinity chromatography
(Bjorck et al., 1984).
RESULTS AND DISCUSSION
Hapten Syn th esis Con sider ation s. Two approaches
were used in hapten synthesis. The first approach
(using haptens derived from deltamethrin, cyper-
methrin, and λ-cyhalothrin) utilized various spacer
attachment points to obtain antibodies with different
selectivities (i.e., attachment site heterology), and the
second approach was to utilize different linking groups
in the conjugation to an enzyme to reduce the affinity
of the antibodies for the spacer arm in immunoassays.
Three series of haptens were synthesized with spacer
attachment (1) at the aromatic moiety, (2) through the
middle of the molecule, and (3) at the cyclopropane
moiety. This approach was attempted to offset different
regions of the molecules from the coupling point; it is
likely that different conformations were also presented
to the immune system since the target pyrethroids are
not planar molecules. In addition, the reaction condi-
tions used would possibly lead to isomerization during
the synthesis; therefore, syntheses were carried out

Hapten Design for Pyrethroid Immunoassays
J. Agric. Food Chem., Vol. 46, No. 2, 1998 527
using racemic mixtures without identification or sepa-
ration of each stereoisomer. The active esters were
relatively insoluble in the N,N′-dimethylformamide-
buffer coupling solution, and the measured coupling
densities with carrier proteins and peroxidase were
relatively low compared with those of other conjugates
[e.g. McAdam et al. (1992)], even using active esters at
a 40 molar excess. The hydrophobic nature of these
haptens probably provides less interaction of the active
ester and the amino groups on lysine residues of the
carrier protein.
placement of a cyano group with the carboxylic acid
group may influence the affinity of the antibody. The
cyano group of a cyanazine hapten was shown to be
essential to raise antibodies specific for cyanazine,
suggesting this group was a strong haptenic determi-
nant (Lawruk et al., 1993). Hydrolysis of deltamethrin
and R-cypermethrin (single isomer pyrethroids) and
λ-cyhalothrin (four isomers) produced acids with one (for
deltamethrin and R-cypermethrin) and four (for λ-cy-
halothrin) stereoisomers, according to proton and carbon
NMR data, presumably similar to the original com-
pounds. Esterification of 3-phenoxymandelic acid with
pyrethric acid chloride produced a pyrethroid analogue
containing a carboxylic acid instead of a cyano substitu-
ent, allowing the formation of an amide derivative with
tert-butyl-â-alanine. Three different pyrethric acids,
deltamethric acid, permethric acid, and cyhalothric acid,
were employed here to obtain structurally different
haptens on the cyclopropane moiety. The esterification
resulted in a number of isomers (according to NMR
data), and the combined isomers were used in the
succeeding step without an attempt to identify the
stereochemistry of these isomers.
Cou p lin g a t th e Cyclop r op a n e Moiety. Coupling
via the cyclopropane moiety was achieved by oxidation
of the dimethylvinyl group. This approach enabled the
aromatic moiety of pyrethroid to be offset from the
coupling point but still retained the cyclopropane ring.
An immunoassay developed using this synthetic prin-
ciple provided sensitive detection of bioresmethrin in
grain (Hill et al., 1993). In the present synthetic
scheme, a different protecting group (tert-butyl ester)
and a racemic mixture of chrysanthemic acid were used
(Figure 2). The use of 2-(trimethylsilyl)ethanol was
initially attempted, but deprotection of the trimethyl-
silyl group with tetraethylammonium fluoride could not
be achieved without loss of the cyano group.
Cou p lin g a t th e Ar om a tic Moiety. Initial at-
tempts to introduce a spacer arm at the aromatic moiety
of the pyrethroid were through a nitro group introduced
on the para position of the phenoxybenzyl group. This
approach would provide a hapten with the cyclopropane
moiety distal to the point of coupling, retaining the full
pyrethroid structure. The synthetic route approached
involved a multiple-step reaction, similar to that of
Demoute et al. (1984), and was initiated by a reaction
of p-nitrophenol and m-bromophenyl-1,3-dioxalane to
form p-nitrophenoxybenzyl-1,3-dioxalane. A succinyl
linker was formed by esterification of the reduced 1,3-
dioxalane derivative with succinic anhydride. The
deprotection of the 1,3-dioxalane group was achieved
following the protection of the carboxylic acid of the
succinyl linker with a tert-butyl group, and the resulting
aldehyde was reacted with NaCN and water to form a
cyanohydrin derivative. However, the esterification of
the cyanohydrin derivative with deltamethric acid
chloride to form a succinyl derivative of deltamethrin
could not be achieved. Thus, various approaches were
attempted, including a formation of a succinyl derivative
after the esterification of deltamethric acid chloride with
p-nitrophenoxybenzyl derivative. The reaction of the
amino group with succinic anhydride, however, could
not be achieved without a simultaneous removal of the
cyano group. Use of 2-(trimethylsilyl)ethyl succinate
instead of succinic anhydride resulted in a loss of a
spacer arm through hydrolysis when esterification was
performed with deltamethric acid. The next attempt
was to form R-cyano-3-benzyl(2,2-dibromovinyl)-2,2-
dimethylcyclopropanecarboxylate, and a spacer arm was
to attach at the meta position of the aromatic moiety
via reduction to produce hydroxy derivative. This
approach, however, produced multiple products with
very low yields, while a reaction of 4-(benzyloxy)phenol
and 2-(3-bromophenyl)-1,3-dioxalane using Ullmann
Syn th esis of Meta bolite An a logu es. Haptens us-
ing the pyrethric acids of deltamethrin, cypermethrin,
and λ-cyhalothrin and the phenoxybenzyl moieties,
namely 3-phenoxybenzoic acid and 3-phenoxybenzalde-
hyde cyanohydrin, were also synthesized. The reaction
of each pyrethric acid (deltamethric, cypermethric, and
cyhalothric acids) with tert-butyl 3-aminopropanoate
yielded a tert-butyl ester. The resulting product was a
racemic mixture even though a single isomer of each
deltamethric and cypermethric acid was used, indicating
the acid had been isomerized.
synthesis resulted in varying degrees of success.
A
In itia l Scr een in g of An tibod y/En zym e Con ju -
ga te Com bin a tion s. Each of the haptens was suc-
cessfully coupled to carrier proteins and HRP, with
coupling ratios as follows: (1) for DEL (compound 14),
6.0 mol of hapten/mol of OA, 14.3 mol of hapten/mol of
KLH, and 3.8 mol of hapten/mol of HRP; (2) for CYP
(compound 15), 3.3 mol of hapten/mol of OA, 4.5 mol of
hapten/mol of KLH, and 2.3 mol of hapten/mol of HRP;
(3) for CYH (compound 16), 4.2 mol of hapten/mol of
OA, 3.9 mol of hapten/mol of KLH, and 3.6 mol of
hapten/mol of HRP; (4) for LHDEL (compound 24), 5.7
mol of hapten/mol of OA, 8.7 mol of hapten/mol of KLH,
and 4.7 mol of hapten/mol of HRP; (5) for LHCYP
(compound 25), 5.3 mol of hapten/mol of OA, 7.6 mol of
hapten/mol of KLH, and 6.2 mol of hapten/mol of HRP;
(6) for LHCYH (compound 26), 5.8 mol of hapten/mol
of OA, 3.2 mol of hapten/mol of KLH, and 1.5 mol of
hapten/mol of HRP; (7) for FULPYR (compound 20), 3.9
mol of hapten/mol of OA and 2.7 mol of hapten/mol of
simple two-step reaction was, therefore, used to intro-
duce a spacer on the aromatic moiety. Deltamethrin
hemisuccinate was prepared by the esterification of
deltamethrin alcohol with succinic anhydride. The
deltamethrin alcohols (at the 2- and 4-positions) were
prepared using lead(IV) trifluoroacetic acid (LTTFA)
and have been assumed to maintain the original ster-
eochemistry (Zhang and Scott, 1994). As noted by these
authors, the yields of the alcohols produced in this
manner were very poor but, in our hands, were suf-
ficient to develop haptens for immunization and enzyme
conjugation.
Con ju ga tion th r ou gh th e Mid d le of th e Mol-
ecu le. This approach produced haptens retaining most
of the structure of the original pesticide, except for the
cyano group (Figure 1). Antibodies raised against these
haptens would be expected to recognize both the cyclo-
propane moiety and the phenoxybenzyl moiety. The dis-

528 J. Agric. Food Chem., Vol. 46, No. 2, 1998
Lee et al.
Ta ble 1. In h ibition by Un isom er ized Delta m eth r in (10 µg/L) of Va r iou s An tibod y/Con ju ga te Com bin a tion sa
antibody
enzyme conjugate
DEL
CYP
CYH
PBA
LHDEL
LHCYP
LHCYH
4-OHDEL
DEL
CYP
CYH
PBA
*/*
*/*
*/**
-/*
-/**
*/*
*/*
*/-
*/*
-/*
*/*
**/**
*/***
*/*
-/*
-/*
-/*
*/**
-/-
*/*
-/*
-/*
*/*
**/*
-/*
-/*
*/*
*/*
*/*
*/*
*/-
*/-
-/-
*/-
*/-
-/-
-/-
-/-
-/-
-/-
-/*
*/*
-/*
*/-
-/-
-/-
-/-
-/-
**/-
**/*
-/*
-/*
*/*
-
-
-
-
-
-
-
-
-
-
*
*/-
-/*
-/*
*/*
-/*
-/*
-/-
-/*
*/**
*/*
-/-
*/*
-/-
*/*
*/-
-/*
*/-
-/-
*/-
*/-
-/*
*/-
-/*
*/*
-/-
-/-
*/-
-/-
-/-
-/-
-/-
*/-
**/-
*/*
-/-
-/-
PBCY
LHDEL
LHCYP
LHCYH
FULPYR
4OHDEL
2OHDEL
LHDEL-BULKY
PBA-BULKY
CYP-BULKY
*/*
*/*
*/*
*/*
-/*
*/*
*
-
-
*/-
*/*
*/*
a
- denotes <10% inhibition, * denotes 10-25% inhibition, ** denotes 26-50% inhibition, *** denotes 51-75% inhibition, and ****
denotes 76-100% inhibition of antibody binding. Asterisks on the left-hand side of the slash represent inhibition values for antibody
raised against KLH conjugates and on the right-hand side for OA conjugates. The concentrations of enzyme conjugates used were optimized
in preliminary experiments to provide an OD of 0.8-1.2. Data shown are means of quadruplicate determinations.
Ta ble 2. In h ibition by Isom er ized Delta m eth r in (10 µg/L) of Va r iou s An tibod y/Con ju ga te Com bin a tion sa
antibody
enzyme conjugate
DEL
CYP
CYH
PBA
LHDEL
LHCYP
LHCYH
4-OHDEL
DEL
CYP
CYH
PBA
*/*
****/***
***/**
**/**
**/**
**/-
**/**
**/**
*/*
***/*
****/*
*/*
**/*
*/*
***/**
**/**
*/*
*/*
*/*
**/-
***/*
**/*
**/*
*/**
-/**
*/*
*/**
***/***
*/*
*
**
*
-
-
-
-
-
*
*
*
*
-
-
*/**
*/*
**/***
*/**
***/-
*/***
*/**
*/**
*/*
**/**
-/*
-/**
*/**
**/***
*/*
*/**
**/*
-/*
*/**
*/**
*/**
**/**
*/*
-/*
-/-
*/-
*/-
-/-
-/-
-/-
*/-
**/-
*/*
**/*
-/-
PBCY
LHDEL
LHCYP
LHCYH
FULPYR
4OHDEL
2OHDEL
LHDEL-BULKY
PBA-BULKY
CYP-BULKY
-/**
***/*
***/-
*/*
*/*
**/**
**/*
**/**
***/**
*/*
*/-
-/*
-/*
-/*
**/*
-/*
***/**
**/**
*/**
***/***
*/*
-/**
**/**
*/*
**/*
**/**
*/*
**/**
*/*
-/**
*/*
-/-
-/-
*/*
a
Other details as for Table 1.
KLH (FULPYR-HRP precipitated); (8) for 4OHDEL
(compound 4), 4.5 mol of hapten/mol of OA, 4.7 mol of
hapten/mol of KLH, and 6.6 mol of hapten/mol of HRP;
(9) for PBA, 4.4 mol of hapten/mol of OA, 3.1 mol of
hapten/mol of KLH, and 7.5 mol of hapten/mol of HRP;
(10) for PBCY (compound 28), 5.9 mol of hapten/mol of
OA, 9.1 mol of hapten/mol of KLH, and 6.2 mol of
hapten/mol of HRP; (11) for 2OHDEL (compound 3), 8.2
mol of hapten/mol of HRP; (12) for LHDEL-BULKY
(compound 30), 1.5 mol of hapten/mol of HRP; (13) for
CYP-BULKY (compound 34), 2.6 mol of hapten/mol of
HRP; and (14) PBA-BULKY (compound 32), 1.4 mol of
hapten/mol of OA, 2.2 mol of hapten/mol of KLH, and
1.7 mol of hapten/mol of HRP.
The concentrations of enzyme conjugates were opti-
mized in initial experiments to provide assay color
development between 0.8 and 1.2 units. Fifteen anti-
sera prepared using 8 haptens were each screened with
14 peroxidase conjugates, providing 210 assays for
evaluation (Tables 1-4). To select antibody/enzyme
conjugate combinations that provided relatively high
sensitivity for one or more of the target compounds,
inhibition values for deltamethrin at 10 µg/L (Table 1)
and 1 mg/L were initially established, using the lowest
conjugate dilutions that provided an OD of 0.8. Al-
though a large proportion of antibody/conjugate combi-
nations exhibited 30-70% inhibition for deltamethrin
at 1 mg/L, only a few combinations were inhibited by
>25% at 10 µg/L. Isomerization of pyrethroids has been
noted as a part of their chemical and photochemical
transformations (Ruzo et al., 1977; Maguire, 1990;
Perschke and Hussain, 1992). The isomerization of
deltamethrin in solvents such as methanol, acetone, and
acetonitrile is thought to occur through a proton ex-
change with the solvent at the R-carbon of the benzyl
group (Ruzo et al., 1977; Perschke and Hussain, 1992).
It occurs only with the type II pyrethroids, such as
deltamethrin, cyfluthrin, cypermethrin, and λ-cyhalo-
thrin (Perschke and Hussain, 1992). Isomerization can
be slowed by acidification and occurs only in solvents
that hydrogen bond the proton at the R-carbon (Per-
schke and Hussain, 1992). Since it was anticipated that
hydroxide ions would accelerate proton exchange with
solvent, the effects of addition of NaOH to pyrethroid
standards dissolved in methanol were investigated. The
isomerization of pyrethroids in methanol was initially
examined by adding different concentrations of NaOH
in water [to 0.5% (v/v), final] in methanol containing a
1 g/L stock of deltamethrin and incubating for 30 min
prior to preparation of deltamethrin standards in water
(Figure 3A). The sensitivity of the assay increased with
base concentration; presumably the extent of deltame-
thin isomerization in 30 min increased with base
concentration (Figure 3A). The isomerization reached
maximum at 5 mM NaOH. The isomerization of del-
tamethrin occurred almost immediately upon the ad-
dition of NaOH (Figure 3B). The rates of isomerization
of cypermethrin and λ-cyhalothrin were somewhat

Hapten Design for Pyrethroid Immunoassays
J. Agric. Food Chem., Vol. 46, No. 2, 1998 529
Ta ble 3. In h ibition by Isom er ized Cyp er m eth r in (10 µg/L) of Va r iou s An tibod y/Con ju ga te Com bin a tion sa
antibody
enzyme conjugate
DEL
*/*
**/**
*/**
***/**
***/****
-/*
*/*
*/*
***/**
-/*
-/*
**/**
**/****
*/*
CYP
*/**
*/-
**/*
****/****
****/*
-/*
*/*
-/-
**/**
****/***
**/**
***/***
****/****
*/*
CYH
PBA
**/*
**/***
*/**
**/**
*/*
**/**
**/**
**/***
**/**
**/***
***/***
***/**
*/*
LHDEL
LHCYP
LHCYH
4-OHDEL
DEL
CYP
CYH
PBA
*/-
*/-
*/-
-/*
*/-
-/*
-/-
-/-
-/-
-/-
-/*
*/**
*/*
-/*
**/*
-/-
-/-
-/-
-/-
-/-
-/*
-/*
-/*
-/*
*/*
-
*
-/-
-/-
-/-
-/-
*/-
*/-
*/-
-/*
-/-
*/-
**/*
*/*
**/-
*/-
-
-
-
-
-
-
-
-
-
*
**/**
-/**
PBCY
LHDEL
LHCYP
LHCYH
FULPYR
4OHDEL
2OHDEL
LHDEL-BULKY
PBA-BULKY
CYP-BULKY
**/*
**/*
-/-
*/*
**/***
***/***
****/****
***/****
*/*
*/*
*/*
-/*
*/*
-
-
-/-
-/-
*/*
a
Details as for Table 1.
Ta ble 4. In h ibition by Isom er ized Cyh a loth r in (10 µg/L) of Va r iou s An tibod y/ Con ju ga te Com bin a tion sa
antibody
enzyme conjugate
DEL
*/*
**/*
*/**
***/**
****/****
-/-
-/*
*/*
****/**
*/**
*/*
***/**
**/****
*/*
CYP
*/*
*/-
*/-
***/***
***/-
-/-
-/*
-/*
***/*
****/***
****/***
***/****
***/****
*/*
CYH
*/*
**/*
-/*
**/**
*/***
**/-
**/*
*/-
*/*
**/**
***/**
***/***
**/***
*/*
PBA
**/*
**/***
**/***
**/**
LHDEL
LHCYP
LHCYH
4-OHDEL
DEL
CYP
CYH
PBA
*/*
*/**
***/**
*/*
-/-
*/-
-/*
*/*
-/**
-/*
-/-
*/*
*
*
*
-
-
-
-
-
*
*
*
*
-
-
-/-
*/*
*/**
-/**
*/-
-/*
-/**
*/*
**/**
-/-
*/-
*/-
*/-
-/*
-/-
*/-
**/-
*/*
PBCY
LHDEL
LHCYP
LHCYH
FULPYR
4OHDEL
2OHDEL
LHDEL-BULKY
PBA-BULKY
CYP-BULKY
*/**
**/**
**/***
**/***
**/***
***/***
***/***
***/***
*/*
*/*
*/**
-/-
*/**
*/*
*/**
*/*
*/*
-/**
*/*
-/-
-/-
*/*
a
Details as for Table 1.
slower, with maximum sensitivities obtained after 15
and 30 min, respectively, from the addition of NaOH
(data not shown). Using the antibody/conjugate com-
bination described above, there was at least a 20-fold
increase in assay sensitivity after the alkali treatment.
There was a gradual loss of sensitivity on extended
exposure to alkali, probably due to hydrolysis; the loss
of sensitivity was greater for λ-cyhalothrin than cyper-
methrin. Isomerization of 1 g/L stock pyrethroid stan-
dard in methanol was achieved routinely by adding 5
mM NaOH, and isomerized deltamethrin was stored at
-20 °C for up to 4 weeks without an obvious loss of
assay sensitivity, while the other pyrethroids were
treated immediately before use. The increases in assay
sensitivity with alkali treatment were quite marked; for
example, with antibody DEL-OA and conjugate CYP-
HRP (Figure 3) a 40-fold increase in sensitivity was
noted.
Inhibition data for isomerized deltamethrin, cyper-
methrin, and λ-cyhalothrin at 10 µg/L are shown in
Tables 2-4. This screening method enabled the selec-
tion of antibody/enzyme conjugate combinations that
provide high (>40%) inhibition at this concentration of
one or more of these pyrethroids; combinations that
exhibited poorer inhibition were not investigated fur-
ther. Twenty-six antibody/conjugate combinations were
selected for further study on the basis of the assay
sensitivity, dynamic behavior, and specificity for delta-
methrin, cypermethrin, and cyhalothrin. The concen-
trations of conjugate used routinely in selected immu-
noassays to provide color development between 0.8 and
1.2 OD units were as follows: 0.31 µg/mL in the CYP-
OA/LHCYP-HRP combination, 0.01 µg/mL in CYH-
KLH/LHCYP-HRP, 1.6 µg/mL in DEL-OA/LHCYP, 1.6
µg/mL in PBA-OA/LHCYP, 0.03 µg/mL in LHDEL-KLH/
LHCYP-HRP, 0.9 µg/mL in PBA-OA/LHCYH-HRP, 0.05
µg/mL in LHDEL-KLH/LHCYH-HRP, 0.06 µg/mL in
LHDEL-KLH/LHDEL-HRP, 0.06 µg/mL in DEL-OA/
LHDEL-HRP, 0.005 µg/mL in CYH-KLH/LHDEL-HRP,
0.06 µg/mL in CYP-OA/LHDEL-HRP, 0.06 µg/mL in
CYP-KLH/LHDEL-HRP, 0.4 µg/mL in CYP-KLH/CYP-
HRP, 0.04 µg/mL in CYH-KLH/CYP-HRP, 0.4 µg/mL in
DEL-KLH/CYP-HRP, 0.4 µg/mL in DEL-OA/CYP-HRP,
2 µg/mL in PBA-OA/CYP-HRP, 0.8 µg/mL in LHCYP-
KLH/CYP-HRP, 4 µg/mL in CYP-KLH/DEL-HRP, 0.2
µg/mL in CYP-OA/DEL-HRP, 0.05 µg/mL in CYH-KLH/
DEL-HRP, 0.04 µg/mL in LHDEL-KLH/DEL-HRP, 1.2
µg/mL in PBA-OA/CYH-HRP, 4.9 µg/mL in FULPYR-
KLH/4OHDEL, 4.9 µg/mL in DEL-OA/4OHDEL-HRP,
and 0.5 µg/mL in PBA-BULKY-HRP. In general, most
of the combinations showing high inhibition were het-
erologous immunoassays (i.e. different haptens used for
immunization and conjugation to the reporter enzyme).
A few exceptions, such as the LHDEL-KLH/LHDEL-
HRP and CYP-KLH/CYP-HRP combinations, were ob-
served.
An antibody was raised against the 3-phenoxybenzyl
alcohol hapten to investigate its potential use in a
generic method for detection of pyrethroids with a
phenoxybenzyl moiety. Of all the antisera studied in
this paper, these antisera gave the highest titer with
the corresponding haptens in indirect ELISA. They
detected phenoxybenzyl metabolites at relatively low
micrograms per liter concentrations. However, the

530 J. Agric. Food Chem., Vol. 46, No. 2, 1998
Lee et al.
isomerized (Tables 3 and 4) and unisomerized forms
(data not shown) of cypermethrin and cyhalothrin, when
used in combinations with antibodies to DEL, CYP,
CYH, and PBA. However, the concentrations of the
conjugates used in these immunoassays were extremely
high (23-35 µg/mL) and the stability of the conjugates
was poor. Poor stability of conjugates was defined as
an irreversible loss in assay color when the conjugate
is stored for >1 month at 2 mg/mL at 4-6 °C. Poor
stability was also observed for 2OHDEL-HRP and
4OHDEL-HRP. Conjugates based on CYP-BULKY did
not show a sufficient inhibition of color development for
deltamethrin, cypermethrin, and cyhalothrin (unisomer-
ized and isomerized), possibly due to the low coupling
density. On the other hand, the LHDEL-BULKY-HRP
conjugate showed a high inhibition at 10 µg/L in
conjunction with several antibodies but exhibited poor
stability. For this reason some rather sensitive antibody/
enzyme conjugate combinations, such as the CYP or
CYH antibodies with either 2OHDEL-HRP, 4OHDEL-
HRP, or LHDEL-BULKY-HRP, were not studied in
detail. Only the PBA-BULKY enzyme conjugate ap-
peared to be sufficiently stable to extended storage (6
months at 4 °C). Most of the combinations with the
unstable enzyme conjugates were eliminated from the
selection, except for CYH-KLH/PBA-BULKY, DEL-OA/
4OHDEL, and FULPYR-KLH/4OHDEL combinations;
these three combinations gave reasonably reproducible
results. Other combinations with inhibition values
>40% did not show these effects.
Assa y Sen sitivities for Delta m eth r in , Cyp er -
m et h r in , a n d Cyh a lot h r in . Initially, the potential
adsorption of deltamethrin, cypermethrin, and λ-cyha-
lothrin was examined by preparing standard curves in
both water and 10% methanol and incubating in un-
treated glass tubes and in glass tubes treated with 5%
(w/v) poly(ethylene glycol) (PEG; molecular weight
20 000), using the method described by Helmuth et al.
(1983). Standards of each of the three pyrethroids were
diluted to 1 to 500 mg/L and sat in glass tubes for
various times up to 1 h before addition to the antibody-
coated microwell plate and analysis using an antibody
to CYH-KLH and the LHCYP-HRP conjugate. The
adsorption was determined by reduction in the assay
sensitivity obtained as IC50 and IC15 of the standard
curves. No noticeable adsorption was observed for
deltamethrin or cypermethrin, but up to 30% of the
λ-cyhalothrin in both water and 10% methanol was
adsorbed onto the untreated and 15% onto the treated
glass tubes after 60 min of incubation. Thus, although
the PEG treatment reduced the adsorption, the assay
could be performed without this treatment if the pyre-
throid standards were used within 30 min of prepara-
tion, to minimize significant loss of pyrethroid in
standard solutions.
The water solubilities of pyrethroids are in the very
low micrograms per liter range (Lefebvre et al., 1993);
thus, erroneous data could be obtained if pyrethroid was
not fully soluble under the assay conditions. To deter-
mine the greatest deltamethrin concentration at which
standards could be prepared, a series of stocks at
different concentrations in methanol were diluted 1:10
in water to produce standards and assayed in the
ELISA. Stocks of 200 µg/L and below provided the most
sensitive assays, suggesting that deltamethrin remained
in true solution after dilution. These data emphasize
that the solubilities of pyrethroids in aqueous solution
F igu r e 3. Effects of addition of NaOH on sensitivity for
deltamethrin determined after (A) isomerization for 30 min
with NaOH at 0 (0), 0.05 (+), 0.1 (2), 1 ([), 5 (9), and 10 mM
(b) (data shown are final concentrations of NaOH in methanol
containing 0.5% water) and (B) isomerization using 5 mM
NaOH (final) after 0 min (]), 10 min (b), 30 min (9), 1 h ([),
2 h (2), 5 h (+), and 24 h (f). The unisomerized deltamethrin
is indicated by 0. Data were generated using antibody DEL-
OA and CYP-enzyme conjugate and are the means of two
determinations in triplicate. Standard deviations of percent
inhibition did not exceed 10%; error bars are omitted for
clarity.
antibodies did not provide sufficient sensitivity for the
detection of pyrethroid parent compounds containing the
phenoxybenzyl moiety. Wraith et al. (1986) raised
antibody against 3-phenoxybenzoic acid and found that
the antibody exhibited a low sensitivity for both phen-
oxybenzoic acid and cypermethrin (IC50 of 1 mg/L).
However, the phenoxybenzyl derivative coupled to
protein was successfully used as an immobilized antigen
in a sensitive immunoassay for permethrin and pheno-
thrin, providing a lower detection limit of 1.5 µg/L
permethrin (Skerritt et al., 1992).
Enzyme conjugates with haptens containing the phen-
oxybenzyl moiety, such as PBA, PBCY, and PBA-
BULKY, usually showed >40% inhibition for both

Hapten Design for Pyrethroid Immunoassays
J. Agric. Food Chem., Vol. 46, No. 2, 1998 531
Ta ble 5. IC50 Va lu es of Delta m eth r in , Cyp er m eth r in , a n d Cyh a loth r in for Differ en t An tibod y/En zym e Con ju ga te
Com bin a tion s
IC50 (µg/L)
antibody
DEL-OA
DEL-OA
DEL-OA
enzyme conjugate
DELa
DELb
CYPa
CYPb
CYHa
CYHb
LHDEL
LHCYP
CYP
4′OHDEL
4′OHDEL
DEL
LHCYH
LHDEL
LHCYP
DEL
LHDEL
CYP
CYP
PBA-BULKY
DEL
LHDEL
LHCYP
CYP
-
-
130
-
33
25
34
16
4
2
2
3
4
9
4
2
5
4
9
4
2
4
2
2
6
9
8
4
120
5
-
-
-
-
80
-
-
-
-
-
7
-
-
DEL-OA
-
-
-
FULPYR-KLH
LHDEL-KLH
LHDEL-KLH
LHDEL-KLH
LHDEL-KLH
CYP-KLH
CYP-KLH
CYP-KLH
DEL-KLH
CYH-KLH
CYH-KLH
CYH-KLH
CYH-KLH
CYH-KLH
LHCYP-KLH
CYP-OA
210
70
-
160
1000
-
82
10
45
20
18
-
650
-
200
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
8
-
-
150
50
220
230
-
-
-
-
60
320
-
200
31
40
60
13
22
15
30
86
-
-
-
-
-
1000
-
65
50
12
11
70
-
-
250
50
60
180
-
85
70
320
1000
-
-
1000
-
CYP
DEL
-
CYP-OA
CYP-OA
PBA-OA
PBA-OA
PBA-OA
PBA-OA
LHDEL
LHCYP
LHCYH
LHCYP
CYH
-
-
-
16
5
48
10
-
-
-
-
-
7
4
-
3
-
600
-
-
1000
-
-
CYP
-
1000
1000
a
b
Unisomerized compounds. Isomerized compounds. DEL, deltamethrin; CYP, cypermethrin; CYH, cyhalothrin. Dash indicates IC50
>1000 µg/L.
and the adsorption of pesticide onto the container wall
could be important factors affecting the performance of
the immunoassay.
enzyme conjugates derived from phenoxybenzoic acid
were highly sensitive (Tables 1-4). Most of the selected
immunoassays had moderately steep standard curves,
with a >30% difference in inhibition for 10-fold differ-
ence in deltamethrin concentration; only three antibody/
enzyme conjugate combinations showed poorer dynamic
behavior, including FULPYR-KLH/4OHDEL-HRP (22%
difference in inhibition), CYH-KLH/DEL-HRP (23%),
and PBA-OA/LHCYP (28%).
Twenty-six combinations of antibody and enzyme
conjugate were selected on the basis of the inhibition
data for deltamethrin, cypermethrin, λ-cyhalothrin, and
the isomerized forms of these compounds at 10 µg/L.
The results obtained are summarized in Table 5. Each
immunoassay was further characterized by determining
the sensitivity (determined as IC50), limit of detection
(determined as IC15), and dynamic behavior (difference
in percentage inhibition between 1 and 10 µg/L) for
isomerized and unisomerized deltamethrin, cyper-
methrin and λ-cyhalothrin. The most sensitive immu-
noassay for deltamethrin was obtained using antibody
to LHCYP-KLH and CYP-HRP conjugate, providing an
IC50 of 2 µg/L and a limit of detection of 0.2 µg/L of the
isomerized deltamethrin, and several other assays had
a similar IC50 but slightly higher limits of detection.
Antibodies raised and conjugates prepared to haptens
containing dihalovinylcyclopropane moieties provided
sensitive immunoassays, suggesting that the antibody
specificity may be directed toward this portion of the
molecule rather than the aromatic groups. One possible
explanation for this would be the greater distance
between the 2′-vinyl carbon of the cyclopropane moiety
to the coupling point than the C3′ of phenoxybenzyl
group as determined from molecular modeling of the
hapten coupled through the middle of the molecule,
providing a greater distance in the former haptens from
the carrier protein, facilitating antibody recognition. On
the other hand, the aromatic part of the pyrethroid plays
an important role in establishing sensitive assays.
First, the antibodies generated to haptens that con-
tained only the dihalovinyl cyclopropane moieties usu-
ally detected the three pyrethroids less sensitively than
those raised to the full synthetic pyrethroid or to
phenoxybenzoic acid. Second, some of the assays using
While several of these immunoassays have high
sensitivity for isomerized deltamethrin, different selec-
tivities were obtained by using different enzyme con-
jugates. For example, when the antibody to DEL-OA
was used with enzyme conjugates based upon LHDEL,
LHCYP, and 4OHDEL, immunoassays were selective
for isomerized deltamethrin, which largely contains
insecticidally inactive isomers. However, when CYP-
HRP was used with this antibody, the assay detected
both isomerized deltamethrin and cyhalothrin but only
relatively poorly detected cypermethrin, even though a
derivative of this compound was used in the conjugate.
The poor ability of cypermethrin to displace antibody
binding may relate to the halogen substituents being
smaller that those on the other pyrethroids. Another
example is the antibody raised against PBA-OA. The
immunoassays using this antibody typically provided a
broad selectivity, but the degree of the cross-reaction
for each compound varied with different enzyme con-
jugates. The PBA-OA antisera/LHCYP (cyclopropane
moiety of cyhalothrin)-HRP combination enabled the
detection of deltamethrin, cypermethrin, and λ-cyhalo-
thrin (in the isomerized forms) at similar sensitivities.
The cross-reaction of cyhalothrin decreased to 50%
(compared with the other conjugates) when LHCYH-
HRP was used. When an enzyme conjugate using a
hapten derived from the entire pyrethroid molecule was
used in a combination with antibody to PBA-OA, the
assay sensitivity increased significantly. In addition,

532 J. Agric. Food Chem., Vol. 46, No. 2, 1998
Lee et al.
Ta ble 6. IC50 Va lu es for Delta m eth r in Isom er s
IC50 (µg/L)
DM4 DM1′
antibody
DEL-OA
DEL-KLH
CYP-OA
CYH-KLH
CYH-KLH
CYH-KLH
CYH-KLH
LHDEL-KLH
LHDEL-KLH
LHDEL-KLH
LHCYP-KLH
FULPYR-KLH
enzyme conjugate
DM1
DM2
DM3
DM2′
DM3′
DM4′
CYP
CYP
DEL
52
60
480
140
73
120
175
130
170
150
160
155
11
5
10
16
16
3
29
40
25
21
4
-
-
-
-
-
-
-
-
-
-
-
-
420
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
LHDEL
LHCYP
CYP
PBA-BULKY
DEL
LHDEL
LHCYP
CYP
700
190
950
825
-
-
510
-
4′OHDEL
55
a
Deltamethrin isomers are as follows: DM1, 1R cis RS; DM1′, 1S cis RR; DM2, 1R cis RR; DM2′, 1S cis RS; DM3, 1R trans RS; DM3′,
1S trans RR; DM4, 1R trans RR; DM4′, 1S trans RS. The stereochemistry of isomers is indicated in order of geometry of the double bond
of the 3-substituent in the cyclopropane ring, configuration at the 1- and 3-positions in the cyclopropane ring, and configuration of the
cyano-bearing carbon of the alcohol component. Dash indicates IC50 >1000 µg/L.
with the use of CYP-HRP the cross-reaction for cyper-
methrin increased, while cross-reaction for cyhalothrin
decreased. The cross-reaction for cyhalothrin increased
when CYH-HRP was used in the immunoassay. Thus,
both the specificity and the sensitivity of the immu-
noassay can be manipulated by the use of different
enzyme conjugates for a given antiserum.
described in Figure 1 used the same general approach
as the bioresmethrin hapten synthesis, although chrys-
anthemic acid was used for re-esterification with the
aromatic moiety. Even though ozonolysis of the vinylic
side chain of the pyrethroid to produce a carboxylic acid,
followed by the direct coupling of the acid to a carrier
protein, was successful for synthesis of phenothrin
haptens (Stanker et al., 1989), this approach was not
successful for dihalovinyl pyrethroids.
Ster eoselectivity of An tibod ies to Delta m eth r in
Isom er s. The stereoselectivity of the antibodies (using
12 different antibody/enzyme conjugate combinations)
was determined by comparing standard inhibition curves
for eight deltamethrin isomers (Table 6). Only two of
the eight deltamethrin isomers, DM1 (the insecticidally
active isomer of deltamethrin, the RS, 1R cis isomer)
and DM2 (the RR, 1R cis isomer), were detected, with
DM2 (the RR, 1R trans isomer) being detected more
sensitively than DM1. Some antibodies were also
capable of detecting DM4, but at very high concentra-
tions. These three isomers have in common the R
configuration at C1 and C3 of the cyclopropane ring,
suggesting that the antibodies may be specific for 1R,3R
configuration at the cyclopropane moiety. This degree
of isomer specificity was unexpected as antibodies were
raised against mixtures of isomers. A possible explana-
tion is that isomerization of the hapten in the immu-
nogen occurred in vivo and/or the biodegradation rates
of each isomer differ in vivo. Indeed, the RS, 1S cis
configuration is unstable at room temperature (Roussel
Uclaf, unpublished data), and the antibody response to
freshly prepared dilutions of the isomers with the S
configuration at the C1 and C3 of cyclopropane ring was
minimal.
Demoute et al. (1986), in a patent application, were
the first to attempt hapten synthesis for a cyanopyre-
throid, deltamethrin, but no immunoassay was de-
scribed. The approach was to introduce a linker on the
3-phenoxybenzyl end of the molecule. Their synthesis
involved six steps. We utilized a much simpler ap-
proach based on direct derivatization of deltamethrin
with lead(IV) trifluoroacetate to produce a phenol
derivative for subsequent coupling. In developing the
immunoassay for the human metabolites of fenvalerate
and fenpropathrin, Wengatz et al. (1994) also synthe-
sized the pyrethroid analogues with an amine substitu-
tion on the aromatic group. Attempts to synthesize
haptens using the pyrethroid metabolites were first
reported by Wraith et al. (1986). The antibody was
produced to a protein conjugate prepared via the three-
carbon chain length spacer arm with 3-phenoxybenzoic
acid and dichlorovinyldimethylcyclopropanecarboxylic
acid. The antibody recognized 3-phenoxybenzoic acid
and cypermethrin but not dichlorovinylcyclopropanecar-
boxylic acid. The assay was applied to soil and water
even though the limit of detection was very high, with
an IC50 of 1 mg/L. Direct coupling of (1R)-cis-permethric
acid and (1R)-trans-permethric acid to the carrier
proteins via carbodiimide activation of the carboxylic
acids was reported by Pullen and Hock (1995a,b) in the
course of our study. Both polyclonal and monoclonal
antibodies were generated, showing different cross-
reactions. The monoclonal antibody (Pullen and Hock,
1995a) detected the type I pyrethroids allethrin, bioal-
lethrin, S-bioallethrin, permethrin, bioresmethrin, and
pyrethrin, suggesting the dimethylcyclopropane moiety
was a haptenic determinant group. Allethrin was
detected with the most sensitivity and a limit of detec-
tion of 1 µg/L. The polyclonal antibody (Pullen and
Hock, 1995b) detected permethrin, cyfluthrin, cyper-
methrin, S-bioallethrin, and trans-permethric acid with
a detection limit of ∼0.4 µg/L. The haptenic determi-
nant group appeared to be (1R)-trans-dichlorovinyldi-
GENERAL DISCUSSION
In this study, we have attempted to evaluate system-
atically strategies for the development of haptens for
the type II pyrethroids, utilizing a range of hapten
coupling positions. The hapten synthesis approaches
combined both novel strategies and adapted concepts
that had been successful for development of pyrethroid
haptens in previously reported studies. The develop-
ment of haptens based on an entire pyrethroid molecule
for immunogens has been reported for the type I
pyrethroids, phenothrin/permethrin (Stanker et al.,
1989), and bioresmethrin (Hill et al., 1993). In contrast
to their approach, we found only the full pyrethroid
hapten (coupled through the vinyl group) in the HRP
conjugate to be useful in the assays. The synthesis

Hapten Design for Pyrethroid Immunoassays
J. Agric. Food Chem., Vol. 46, No. 2, 1998 533
methylcyclopropane moiety. Both antibodies, however,
were unable to detect deltamethrin or other type II
pyrethroids containing different halogen atoms, such as
λ-cyhalothrin. Bonwick et al. (1994) have also reported
the synthesis of haptens based on permethrin hydrolysis
products, but in this case using either 6-aminohexanoic
acid or 4-aminobutanoic acid spacer arms to link the
permethric and phenoxybenzoic acids to protein carriers.
The best assay using, the latter hapten, was not
particularly sensitive (IC50 of 1 mg/L).
28; 2OHDEL, compound 3; LHDEL-BULKY, compound
30; CYP-BULKY, compound 34; PBA-BULKY, com-
pound 32.
ACKNOWLEDGMENT
We thank Mr. A. Rigg for assistance with the syn-
thesis of haptens DEL and PBCY. We are grateful to
Dr. M. H. Black, Director of Research and Development,
AgrEvo Environmental Health Ltd., Berkamsted, Herts,
U.K., for provision of deltamethrin isomers. We ac-
knowledge the advice and assistance of R. Mayadunne
and Professors L. Mander and J . Elix (Australian
National University) and Dr. J . E. Nemorin (University
of Sydney).
The syntheses we undertook provided a series of
immunogens and enzyme conjugates differing in the
structures of the haptens and the spacer arms, and in
the coupling point, thus exposing different portions of
pyrethroid for antibody recognition. The resulting
antibodies provided more than two dozen useful immu-
noassays with different selectivities for deltamethrin,
cypermethrin, and λ-cyhalothrin in both unisomerized
and isomerized forms. Isomerization of deltamethrin,
cypermethrin, and λ-cyhalothrin was achieved by simply
adding 5 mM NaOH to the stock standard in methanol,
and the rate of isomerization was in the order delta-
methrin > cypermethrin > λ-cyhalothrin, with delta-
methrin being the most tolerant to hydrolysis. In
general, the sensitivity was greater for isomerized
compounds than for unisomerized compounds. The
most sensitive immunoassay enabled detection of del-
tamethrin as low as 0.2 µg/L (isomerized). Assay
specificity was able to be manipulated by varying the
haptens coupled to the detection enzyme. The antibody
specificities were usually determined by the portion
distal to the carrier protein. That is, antibodies raised
to the aromatic end of pyrethroids were able to detect
compounds with this group, such as cypermethrin,
deltamethrin, λ-cyhalothrin, permethrin, esfenvalerate,
fluvalinate, and phenothrin, and antibodies generated
from the cyclopropane end generally detected com-
pounds with the cyclopropane moiety, such as delta-
methrin, cypermethrin, λ-cyhalothrin, permethrin,
tetramethrin, bioallethrin, allethrin, S-bioallethrin, cy-
fluthrin, and bifenthrin [data in the accompanying
paper, Lee et al. (1998)]. This initial development
provides a basis for further selection of the most useful
immunoassays and a complete characterization of assay
sensitivity, specificity, and precision. The specificity
properties and validation of the performance of these
assays using spiked water, soil, and grain samples and
relationships between immunoassay data with GC/MS
analyses for spiked and field samples are described in
the following paper (Lee et al., 1998).
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Received for review May 23, 1997. Revised manuscript
received November 3, 1997. Accepted November 10, 1997. We
are grateful to the Australian Cotton Research and Develop-
ment Corporation for financial support.
J F970438R